Posters

Transposons & Epigenetics

P1: A role for RdDM in susceptibility to paramutation at the maize BOOSTER1 locus

Transposons & Epigenetics Kevin Peek (Graduate Student)

Peek, Kevin1
Lauss, Kathrin1
Bader, Rechien1
Lee, Tzuu-fen2
Meyers, Blake2 3
Scheuermann, Daniela4
Weltmeier, Fridtjof4
Kolkmeyer, Bastian4
Stam, Maike1

1Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098XH Amsterdam, The Netherlands
2Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711
3Biology Department, UC Davis, Davis, CA 95616, USA
4KWS SAAT SE & Co. KGaA, Einbeck, 37574 Einbeck, Germany

Paramutation is defined as in trans ­­communication between alleles, whereby a silent epigenetic state is heritably transferred to an active (epi)allele. When the active copy is silenced, it becomes able to induce silencing of other active copies (secondary paramutation). Paramutation of the BOOSTER1 (b1) locus in maize (Zea mays) is one the best studied systems. B1 encodes a transcription factor in the anthocyanin biosynthesis pathway. Two epialleles participate in b1 paramutation: the lowly expressed B’ allele and the highly expressed B-Intense (B-I) allele1. A seven-times repeated 853bp sequence located about 100kb upstream of b1 is required for paramutation and high expression of b12. The region required for paramutation was mapped to the 5’ half of the repeat unit3. Based on DNA methylation levels, two differentially methylated regions (DMR1 and DMR2) were identified in each repeat unit4. At the inactive B’ epiallele, DMR1 and DMR2 are highly methylated in CG and CHG context, whereby low levels of 24nt siRNAs levels are produced from DMR1. At the active B-I epiallele, DMR1 is unmethylated while DMR2 shows high CG, CHG and CHH methylation and produces relatively high 24nt siRNAs levels4. Counterintuitively, these findings implicate the presence of the RNA-directed DNA Methylation (RdDM) machinery on DMR2 of the B-I epiallele. We hypothesize that the RdDM machinery on DMR2 of B-I is required for susceptibility to paramutation. An adapted RRBS method5 provided preliminary indications that upon paramutation of B-I, DNA methylation is spreading from DMR2 into DMR1. To further explore our hypothesis, we are currently investigating DNA methylation patterns, 24nt siRNA production, and H3K9me and H3K27me deposition at the b1 repeats of B-I, B’, and their F1 in several developmental stages. Furthermore, we are using a transgenic approach to define the roles of DMR1 and DMR2 in paramutation at the b1 locus. 1. Coe, E. H. (1959). 2. Stam, M. et al. (2002). 3. Belele, C. L. et al. (2013). 4. Lauss, K. PhD thesis (2017). 5. Edelmann, S. & Scholten (2018).

P2: An epigenetic basis for inbreeding depression in maize

Transposons & Epigenetics Z Jeffrey Chen (Principal Investigator)

Han, Tongwen2 3
Wang, Fang2 3
Chen, Z Jeffrey1

1The University of Texas at Austin
2Nanjing Agricultural University
3Shandong Agricultural University

Inbreeding depression is a widespread across plant and animal kingdom and may arise from exposure of deleterious alleles and/or loss of overdominant alleles resulting from increased homozygosity. However, these genetic models cannot fully explain the phenomenon. For example, yield in maize inbred lines continues to decline even after 10 generations of self-pollination. Here we present an epigenetic basis of inbreeding depression in maize. Teosinte branched1/Cycloidea/Proliferating-cell-factor (TCP) transcription factors control plant growth and development. During successive inbreeding among highly inbred lines, thousands of genomic regions across TCP-binding sites (TBS) are hypermethylated through the H3K9me2-mediated pathway. These hypermethylated regions are accompanied by decreased chromatin accessibility, increased levels of the repressive histone marks H3K27me2 and H3K27me3, and reduced binding-affinity of maize TCP proteins to TBS. Consequently, hundreds of TCP-target genes involved in mitochondrion, chloroplast, and ribosome functions are downregulated, leading to reduced growth vigor. Conversely, random matting can reverse the corresponding methylation sites and TCP-target gene expression, restoring growth vigor. These results support a unique role of reversible epigenetic modifications in inbreeding depression in maize.

P4: Characterization of two epigenetically regulated ABI3-VP1 transcription factors in Zea mays (maize)

Transposons & Epigenetics Carly Blair (Graduate Student)

Blair, Carly J1
Riboldi, Lucas B1
Madzima, Thelma F1

1Michigan State University; East Lansing, Michigan, USA 48824

Transcriptional regulation via epigenetic mechanisms is critical for proper plant function and development. In plants, the RNA-directed DNA methylation (RdDM) pathway has been shown to maintain genomic integrity by transcriptionally silencing transposable elements (TEs) and genes via cytosine methylation. In maize, mediator of paramutation 1 (mop1) is a RNA-dependent RNA polymerase that is crucial for RdDM pathway function, and mop1-1 mutant plants are deficient in cytosine methylation and transcriptional gene silencing. Large-scale disruption of the RdDM pathway results in a variety of developmental defects, and baseline dysregulation of several genes including two transcription factor (TF) paralogs designated transcriptional gene silencing 2 (tgs2) a and b. These proteins belong to the ABI3-VP1 TF family; characterized by involvement in seed maturation and response to the plant hormone abscisic acid (ABA). However, tgs2a and tgs2b show differential regulation in mop1-1 plants without exogenous ABA treatments. To characterize the functions of these two proximal genes, upstream regulators and predicted targets were identified using the Maize Gene Regulatory Network. Most of the resulting regulators and targets were unique between the two TFs, suggesting these genes have evolved specialized tissue-specific function. This idea is additionally supported by preliminary locus-specific bisulfite sequencing data showing differential RdDM-dependent methylation patterns at the transcriptional start sites of both TFs. To further characterize the function of these TFs, we identified UniformMu insertion stocks with insertions in 5’ untranslated region of tgs2b. Preliminary analyses suggest that tgs2b plays a role in early developmental patterning and organ formation. Notably, tgs2b mutants exhibit abnormal elongation of the ear shank after pollination, particularly in the internode regions, while overall ear size remains unaffected. Characterization of tgs2a and tgs2b is expected to provide new insights into how epigenetic regulation of TFs contributes to developmental gene networks in maize.

P5: Characterizing chromatin accessibility of long terminal repeat retrotransposons in response to cold stress in maize

Transposons & Epigenetics Carmen Rodriguez (Graduate Student)

Rodriguez, Carmen M1
Gooden, Caleb1
Li, Xingli1
Ou, Shujun1

1Ohio State University; Aronoff Laboratory 318 West 12th ave.; Columbus, OH, 43016

Cold stress at the seedling stage can reduce maize yield and quality by disrupting critical physiological processes, including photosynthesis, cell membrane integrity, and nutrient uptake. As global temperatures fluctuate and extreme weather events become more frequent, understanding how crops like maize respond to environmental stress is crucial for ensuring sustainable food production. Long terminal repeat retrotransposons (LTR-RTs) make up a large portion of the maize genome and have been shown to influence gene expressions and epigenetic modifications through chromatin remodeling. However, their chromatin accessibility dynamics in regulating plant responses to cold stress are not yet fully understood. A major challenge in accessing the chromatin status of LTR-RTs is their repetitive nature, preventing accurate mapping and analyzing using short-read sequencing methods. To address this, we are adapting the new Fiber-seq technology for mapping chromatin accessibility. To date, we have successfully isolated high-quality nuclei from maize seedlings and identified an LTR family for focused investigation. Ongoing efforts include scaling up nuclei isolation to meet experimental requirements and validating Fiber-seq labeling of chromatin accessibility in these nuclei. This research provides the groundwork for understanding chromatin remodeling of LTR-RTs in maize cold stress responses. Future directions will explore the functional impact of LTR-RTs for their regulatory potential on cold-responsive genes.

P6: Co-expression analysis unveils the roles of long terminal repeat retrotransposons in maize developmental regulation

Transposons & Epigenetics Abby Sow (Undergraduate Student)

Sow, Abby L1
Gooden, Caleb1
Ou, Shujun1

1Department of Molecular Genetics; The Ohio State University; Columbus, OH, USA 43210

Transposable elements (TEs) are mobile regions of the genome that can influence gene expression. Long Terminal Repeat retrotransposons (LTR-RTs) have regulatory sequences that control their transcription using host machinery and a promoter region. When near a gene, they can regulate its transcription and expression through this ability. Approximately 75% of the maize genome is composed of LTR-RTs, making maize a useful model system for studying LTR-RT-driven regulatory activities in plants.We used a weighted-gene coexpression network analysis (WGCNA) to identify LTR-RTs involved in gene regulatory pathways. This revealed genes that are strongly coexpressed with LTR-RTs and displayed hub-like transcription across multiple tissues. We found a network with connected genes and LTR-RTs that were downregulated in the maize endosperm and upregulated in leaves with gene ontologies associated with photosynthesis and organelle membranes. In this network, the LTR-RT 8065 was designated as the hub, suggesting that LTR-RTs may be responsible for important developmental activities in maize. We are developing a transformation protocol using maize cell cultures to explore how and when LTR-RTs are transcribed. We will look for conserved binding motifs to the LTR-RTs that may be involved in gene regulation. Our results will lead to further characterizations of LTR-RT promoters and investigate meaningful changes to gene regulatory networks. This study of LTR-RTs can be applied to biotechnological and agricultural fields to address agricultural impacts and food insecurity.

P7: Detecting activity of hopscotch family long terminal repeat retrotransposons in Zea mays

Transposons & Epigenetics Patrick Gardner (Graduate Student)

Gardner, Patrick1
Isles, Taylor2
Zhao, Meixia3
Du, Charles1

1Department of Biology; Montclair State University; Montclair, NJ, US 07043
2Department of Biological Sciences; University of Missouri; Columbia, MO, US 65211
3Microbiology & Cell Science Department; University of Florida; Gainesville, FL, US 32611

Long terminal repeat retrotransposons (LTRs) are a group of class 1 transposable elements that copy and reintegrate themselves throughout a genome, proliferating over time. This has resulted in LTRs composing a significant proportion of many eukaryotic genomes, from roughly 8% in humans, to as much as 75% in maize. The majority of LTR copies are degraded, from a combination of improper replication due to reverse-transcriptase’s lack of proofreading functionality, and mutations over many cell replication cycles. Resultingly, most LTRs in a given genome are nonfunctional, to the extent that some families of LTR retrotransposon lack any fully viable copies, and as a result no longer actively propagate. Previous research into retrotransposon-mediated mutations in maize pollen has suggested that the Hopscotch family of LTRs are still active. Additional examination, using whole genome sequencing data from the Du lab at Montclair State University, and the Zhao lab at the University of Florida, strengthens this suggestion, further indicating continued activity of Hopscotch LTRs in maize. This analysis involved mapping sets of paired-end reads to a conserved reference Hopscotch LTR sequence, and the two inbred line reference genomes each individual sequenced plant was a cross of, generating lists of Hopscotch insertion loci in those individuals. These lists were filtered against an index of prior Hopscotch insertion loci in each reference genome, assembled by mapping the reference Hopscotch sequence against both reference genomes at low coverage and moderate identity, limiting the list of mapped insertion loci to putatively novel sites. This process has yielded multiple potentially novel Hopscotch insertions, and while analysis is ongoing, these insertion loci may be validated as novel by performing the same analysis on the sequencing data of both parental plants. Any insertion locus not represented in either parental genome can reasonably be inferred to have occurred in the intervening generation.

P8: Does active DNA demethylation drive epigenetic silencing in pollen?

Transposons & Epigenetics Jonathan Gent (Research Scientist)

Gent, Jonathan I1
Sun, Yirui1

1University of Georgia; Athens, GA, USA 30602

Multiple studies have reported that DNA demethylation by DNA glycosylases (DNGs) in the pollen vegetative nucleus reinforces methylation in the two sperm nuclei. These reports have proposed an elegant though counterintuitive mechanism behind this: Demethylation of transposons allows transcription that then leads to siRNA production, and the siRNAs move into the sperm nuclei to direct silencing of homologous loci. In this way, DNA demethylation exposes potentially pathogenic transposons in cells where they cannot transmit to the next generation while epigenetically silencing them in the germline itself. Here we interrogate this model through our own data from maize and through reanalysis of the original data from rice. The results do not support the original model. In fact, they suggest that demethylation hinders rather than promotes siRNA production and lead us to conclude that DNGs do not drive epigenetic silencing in pollen.

P9: Genome-wide resolution of long terminal repeat retrotransposon promoters and their tissue-specific regulatory programs

Transposons & Epigenetics Caleb Gooden (Graduate Student)

Gooden, Caleb1
Li, Xingli1
Walter, Isabella1
Ou, Shujun1

1Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA 43210

Gene expression is highly regulated to direct early plant growth and respond to environmental cues. Transposable elements (TEs) are significant components in transcription regulation. The TE subclass long terminal-repeat retrotransposons (LTR-RTs) comprise 75% of the maize genome and represent an underexplored population of potential regulatory features contributing to gene expression. The promoter regions of LTR-RTs can act as additional gene regulators through cis-regulatory activities, but they remain uncharacterized due to their sequence repetitiveness and divergence. The U3 subregion of LTRs is hypothesized to contain the promoter, but what informs its boundary with the downstream transcription start site (TSS) remains uncertain. In this study, we developed IsoClassifier and used long-read cDNA sequencing to precisely identify the TSS of transcribed LTR-RTs and define the upstream U3 promoter region. These TSS predictions were significantly supported by the evidence-based annotation from CAGE-seq and Smar2C2-seq. We identified distinct types of LTR-RT transcript species, implicating the production of long non-coding RNA (lncRNA). These lncRNA candidates comprised 72% of total LTR-RT transcripts, the majority having undergone splicing. Further, we characterized LTR-RTs in the B73 genome using in-house and publicly available histone modification, long-read DNA methylation, genomic annotations, and transcriptomic data of several maize tissue types. By developing WindowScrubber to explore promoters of LTR-RTs and genes, we found shared canonical motif compositions such as the TATA box, suggesting a potentially shared evolutionary origin of transcriptional promoters. Weighted gene co-expression analysis revealed that some LTR-RTs potentially served as regulatory hubs for functional genes, which are relevant to photosynthesis and organelle membranes. These data support a model wherein LTR-RTs are transcribed differentially as components of gene expression pathways throughout maize development. Our work resolves features of U3 promoter regions and expands upon the regulatory capacity of LTR-RTs in maize development.

P10: Helitron-mediated expansion of endosperm regulatory binding sites in maize

Transposons & Epigenetics Daniela Barro Trastoy (Postdoc)

Barro Trastoy, Daniela1
Qiu, Yichun1
Meng, Ling2
Wang, Hong2
Köhler, Claudia1

1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
2KWS Gateway Research Center, LLC. BRDG Park at The Danforth Plant Science Center, 1005 N Warson Road, Ste 201, Saint Louis, MO 63132, USA

The endosperm is a defining innovation of flowering plants and a central determinant of seed viability, yield, and nutritional quality. In cereal crops such as maize (Zea mays), the endosperm constitutes the bulk of the seed and represents a major source of calories for human and animal consumption worldwide. Understanding the molecular mechanisms governing endosperm development and its evolutionary origin is therefore of fundamental and applied importance for crop improvement.Recent work from our group supports the hypothesis that Type I MADS-box transcription factors (TFs) played a key role in the emergence and diversification of the endosperm. In Arabidopsis thaliana, the Type I MADS-box TF PHERES1 (PHE1) functions as a master regulator of endosperm development by controlling genes involved in cellular proliferation. Notably, PHE1 DNA-binding motifs overlap with Helitrons, a class of transposable elements, suggesting that transposition-mediated dispersal of regulatory sequences facilitated the wiring of a coordinated endosperm-specific transcriptional network.To evaluate the evolutionary conservation and crop relevance of this mechanism, we focused on the identification and functional characterization of PHE1-like genes in maize, which has abundant and highly specialized endosperm. We investigated whether Helitrons similarly overlap with binding motifs of maize PHE1-like TFs and assessed the expression and target genes of these factors during seed development.Preliminary expression analyses indicate that PHE1-like genes are active in maize endosperm tissues, consistent with patterns observed in Arabidopsis. Importantly, several putative maize target genes are associated with endosperm proliferation and growth. Together, these findings point to an evolutionarily conserved regulatory module underlying endosperm development and highlight PHE1-like transcription factors and their downstream networks as promising targets for maize breeding and yield optimization.

P11: How transposable elements preserve heterozygosity during inbreeding in maize

Transposons & Epigenetics Hailey Buell (Graduate Student)

Buell, Hailey L1
Favela, Alonso1

1University of Arizona; 1401 E University Blvd; Tucson, AZ, United States, 85721

Classical population genetic theory predicts a rapid and predictable loss of heterozygosity during inbreeding. However, empirical studies in both plants and animals frequently observe substantially higher levels of heterozygosity than expected. A pioneer study in Arabidopsis lyrata proposed that this conservation of heterozygosity was largely due to transposable elements (TEs), which act as regions where heterozygosity is both preserved and generated de novo during inbreeding. We tested whether this pattern extends to maize, a species with a TE-rich genome, by analyzing whole-genome sequences from six maize landraces inbred for six generations (single seed descent). Across all landraces, observed heterozygosity exceeded theoretical expectations following inbreeding. Conserved heterozygosity was distributed non-randomly across the genome and was enriched in genes associated with core cellular maintenance, RNA/DNA processing, and stress response. Notably, the majority of heterozygous TEs contained evidence of de novo heterozygosity. Together, these results support a general role for transposable elements in shaping patterns of heterozygosity during inbreeding and suggest that TEs may actively contribute to the maintenance of functional genetic variation in crop genomes. Moving forward, we will leverage these findings to test how variation in TE load and diversity mediates plant stress responses and modulates interactions with the plant microbiome.

P12: Identification of regulatory genes involved in RNA-directed DNA methylation mediated regulation of responses to abiotic stress in Zea mays (maize).

Transposons & Epigenetics Thelma Madzima (Principal Investigator)

Chen, Huan1
Riboldi, Lucas B.1
Madzima, Thelma F.1

1Department of Plant Biology, Michigan State University, East Lansing, MI 48824

Understanding the fundamental mechanisms regulating plant responses to abiotic stress is necessary to create climate resilient crops. We aim to understand how the plant-specific RNA-directed DNA methylation (RdDM) epigenetic pathway facilitates growth, development, and response to environmental stress in Zea mays (maize). The maize mediator of paramutation 1 (mop1) gene encodes an RNA-dependent RNA polymerase (RDRP) required for progression of the RdDM pathway and regulates transcriptional silencing of loci across the genome. As such, the mop1-1 mutation is a valuable resource to characterize RdDM-mediated activity in maize. In plants, the abscisic acid (ABA) hormone is well-characterized for its role in mediating responses to specific abiotic stress stimuli. We used mop1-1 mutants subjected to ABA treatment to identify synergistic targets of epigenetic and abiotic stress-mediated regulatory networks in maize. Gene co-expression network (GCN) modules and gene ontology (GO) enrichment were used to create gene regulatory networks (GRN). We identified ten (10) candidate regulatory genes of which three (3) of these genes are transcription factors (TFs) predicted to regulate different biological processes in a tissue-specific manner. Enriched target genes are involved in root development, responses to high light intensity, heat, protein phosphorylation, regulation of stomata and nucleosome assembly. This data provides insights into the specific biologically processes epigenetically regulated in response to environmental stress. To experimentally validate the regulatory functions of these genes, we have obtained UniformMu insertion stocks (maizegdb.org) and these have been introduced into a mop1-1 background through crossing for epistatic analysis. Together, this study will allow us to genetically dissect the mechanisms of epigenetic regulation in response to abiotic stress, that can be manipulated to develop climate resistant crops.

P13: Single-molecule methylation profiling at the repetitive b1 paramutation locus in maize

Transposons & Epigenetics Juliette Breil-Aubert (Postdoc)

Breil-Aubert, Juliette1
Peek, Kevin1
Bader, Rechien1
Baulcombe, David C2
Stam, Maike1

1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1098 XH, The Netherlands
2Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom

Paramutation is a fascinating epigenetic phenomenon in which one allele induces a heritable change in the expression of another through the transfer of epigenetic silencing information in trans. This often involves changes in DNA methylation and repressive histone modifications (Hövel et al. 2024; Hollick 2017). To understand how and when such silencing is established and maintained, I investigate DNA methylation patterns at single-molecule resolution across specific loci. This level of analysis is particularly challenging in large, repetitive genomes like that of maize, where cost-effective, targeted methylation profiling is essential. Yet no standard method currently enables this. To address this, I refined Cas9-targeted nanopore sequencing (nCATS), a method that uses Cas9 during library preparation to enrich for specific loci. This enables long-read sequencing and direct detection of cytosine methylation using nanopore technology. With this method, I can resolve methylation patterns across challenging regions such as the b1 paramutation locus in maize, which contains seven nearly identical 853bp tandem repeats that are required for paramutation, but also enhancer activity. nCATS allows to distinguish the methylation state of each individual repeat, enabling for the first time the study of paramutation dynamics at single-molecule resolution. We are now applying nCATS to multiple epialleles at the b1 locus, including B′ (paramutated), B-I (paramutable), and F1 individuals during plant development, when paramutation is being established. This will allow us to pinpoint the timing of methylation gains and losses, track how these patterns emerge across repeats, and determine how their establishment aligns with the onset and progression of paramutation.

P14: TEs in the “Metagenome”: A kmer based approach to estimate TE abundance from short reads

Transposons & Epigenetics Natasha Dhamrait (Graduate Student)

Dhamrait, Natasha1
Ross-Ibarra, Jeffrey1

1University of California Davis, Davis CA, USA 95616

Characterizing the diversity and divergence of transposable elements (TEs) in natural populations is logistically difficult. This is due to the computational cost of current tools and the common requirement of comprehensive genomic resources such as long-read sequencing. The latter is especially limiting, even for traditional varieties of maize, where most populations are sequenced with low coverage short reads. Inspired by metagenomics approaches, we have developed a kmer based pipeline to estimate TE abundance across families using only short reads (at a minimum depth of 2.5x) and a pre-curated TE library from a related genome. TE family abundances in the NAM lines estimated with this approach were ~91% correlated with EDTA. In just a few hours, we can estimate patterns of TE abundance, and diversity for more than 300 zea genomes. With this data, we were able to quantify population specific patterns in TEs across the teosintes, increase our understanding of the early geography of domestication, and explore the eco-evo drivers of TE differentiation between closely related populations in Zea. This pipeline increases the feasibility of quantifying TE dynamics in understudied genomes, including those of agricultural weeds or pests, endangered species, or even your favorite maize variety.

Evolution and Population Genetics

P15: BZea: A diverse teosinte introgression population for improving modern maize sustainability

Evolution and Population Genetics Hannah Pil (Graduate Student)

Pil, Hannah D.1 2
Tandukar, Nirwan1 2
Rodríguez Zapata, Fausto1
Escalona Weldt, Carolina1
Insko, Lauren1
Fazler, Zehta1 2
Stokes, Ruthie1
Rumley, Katelyn3
Romay, Cinta4 5
Barnes, Allison C.1 6
Holland, James B.3 6
Gage, Joseph L.3
Buckler, Edward S.4 5 7
Sawers, Ruairidh8
de Jesús Sánchez González, José9
Rellán Álvarez, Rubén1

1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA
2Genetics and Genomics, North Carolina State University, Raleigh, NC, USA
3Department of Crop and Soil Sciences, North Carolina State University, Raleigh, NC, USA
4Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA
5Section of Plant Breeding and Genetics, Cornell University, Ithaca, NY, USA
6USDA-ARS, Plant Science Research Unit, Raleigh, NC, USA
7USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA
8Department of Plant Science, Pennsylvania State University, State College, PA, USA
9Universidad de Guadalajara, Centro Universitario de Ciencias Biológicas y Agropecuarias, Zapopan, Jalisco, Mexico

Teosinte, the wild ancestor of maize, harbors a rich reservoir of interesting traits that have been largely diminished during maize domestication. Recognizing the potential of these unknown alleles, here we present the genotypic and phenotypic characterization of a new teosinte introgression population and preliminary data on experimental applications of this germplasm. Its donor lines consist of 81 georeferenced teosinte accessions from multiple species across the Zea genus, including Zea mays ssp. parviglumis, ssp. huehuetenangensis, ssp. mexicana, Zea diploperennis, and Zea luxurians, with around 2100 total derived lines. Evaluating the impact of teosinte alleles on agronomic traits is inherently difficult due to the differences in photoperiod and growth habitat of maize. This population, “BZea,” addresses this by creating BC₂S₃s with B73, generating derived lines with approximately 12.5 percent of the original teosinte donors. Introgression into B73 allows for the evaluation of teosinte alleles in a maize background in temperate conditions.To characterize this population, we conducted whole-genome sequencing of 1400 derived lines at an average depth of 0.8× and performed 3′ RNA-sequencing on a subset of these lines. Using this population, we are investigating diverse experimental questions. For example, we are exploring nitrogen dynamics by assessing lines derived from Z. diploperennis, a perennial teosinte that we expect to harbor alleles relevant to nutrient recycling. We are further leveraging the population to generate allelic series for several candidate genes, including those associated with nitrogen metabolism and flowering time. In addition to studying allele function, we are also using this resource for QTL mapping to systematically identify genomic regions where teosinte introgressions contribute to phenotypic variation. Together, these efforts aim to uncover the functional impact of teosinte alleles and their potential to enhance maize agronomic traits.

P16: Booster: Cistrome and transcriptome comparative genomics among species with markedly drought tolerance differences

Evolution and Population Genetics Vincenzo Rossi (Principal Investigator)

Banovic Deri, Bojana1
Lanzanova, Chiara1
Tartaglia, Jacopo2
Faccioli, Primetta2
Engelhorn, Julia3
Hartwig, Thomas3
VanBuren, Robert4
Rossi, Vincenzo1

1Council for Agricultural Research and Economics (CREA), Research Center of Cereal and Industrial Crops, Bergamo, Italy
2Council for Agricultural Research and Economics (CREA), Research Center of Genomics and Bioinformatics, Fiorenzuola D’Arda (PC), Italy.
3Heinrich Heine University Düsseldorf, Faculty of Mathematics and Natural Sciences, Institute for Molecular Physiology, Düsseldorf, Germany
4Michigan State University, Plant Resilence Institute, Department of Plant Biology, Department of Plant, Soil & Microbial Sciences, East Lansing, MI, USA

BOOSTER project: https://boosterproject.eu/ is funded by European Union’s Horizon Europe research and innovation program. BOOSTER aims to improve drought tolerance in two cereal crops, maize and teff, using two synergistically strategies: i) a novel and more efficient exploitation of natural genetic variations, specifically focused on the genome-wide functional identification of cis-regulatory elements (CREs) and ii) the development and the study of mode of action of novel biostimulants derived from living organisms (i. e. sea weeds extracts and plant growth promoting rhizobacteria). The project also investigates Eragrostis nindensis, a desiccation-tolerant grass genetically related to teff, with the additional aim to explore the potential for transferring species-specific drought responsive features. MNase-defined cistrome-Occupancy Analysis (MOA-seq) coupled to high throughput mRNA sequencing (mRNA-seq) was used to identify allele- and condition- (well-watered: WW and drought stressed: DS) specific quantitative variations of transcription factor (TF) binding in the CREs of maize and teff F1 hybrids, as well as condition variations in one E. nindensis genotype. The sequences identified, along with previously published data, have been used to perform comparative genomics analyses among maize, teff, and E. nindensis - three species with markedly different levels of drought tolerance - to identify unique or conserved regulatory features. The ultimate goal is to apply genetic strategies, including genome editing, to transfer specific drought tolerance traits from more resilient to more sensitive species. Comparative analyses were conducted with BLASTN, BLASTP, McScanX, OrthoFinder, and Cactus. Integration of these data revealed syntenic orthogroups shared among all three species or between pairs of species, as well as species-specific features associated with drought-responsive gene expression, providing a foundation for developing diverse genetic strategies for drought improvement. This study illustrates the power of combining high-resolution cistrome and transcriptome mapping with comparative genomics to identify molecular features underlying drought tolerance. [VR1]Maximum length: 300 words

P17: Comparative grass genomics reveals explosive genome evolution in maize and its wild relatives

Evolution and Population Genetics Michelle Stitzer (Postdoc)

Stitzer, Michelle C1
Seetharam, Arun S2
Scheben, Armin3
Hsu, Sheng-Kai1
Schulz, Aimee J1
Aubuchon-Elder, Taylor M4
El-Walid, Mohamed1
Ferebee, Taylor H1
Hale, Charles O1
La, Thuy1
Liu, Zong-Yan1
McMorrow, Sarah1
Minx, Patrick4
Phillips, Alyssa R5
Syring, Michael L2
Wrightsman, Travis1
Zhai, Jingjing1
PanAndropogoneae, Germplasm Collaborators6
Other, PanAndropogoneae Collaborators7
Siepel, Adam3
Ross-Ibarra, Jeffrey5
Romay, M Cinta1
Kellogg, Elizabeth A4
Buckler, Edward S1 8
Hufford, Matthew B2

1Cornell University; Ithaca, NY, USA 14853
2Iowa State University, Ames, IA, USA 50011
3Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 11724
4Donald Danforth Plant Science Center, Saint Louis, MO, USA 63132
5University of California, Davis, Davis, CA, USA 95616
6U. Montpellier; Principa College; U. Georgia; NSF; Mahidol U.; Indiana U.; South China Agricultural U.
7Corteva Agriscience, U. Missouri, MaizeGDB
8USDA-ARS, Ithaca, NY, USA 14853

Over the last 20 million years, the Andropogoneae tribe of grasses has evolved to dominate 17% of global land area. Domestication of these grasses in the last 10,000 years has yielded our most productive crops, including maize, sugarcane, and sorghum. The majority of Andropogoneae species, including maize, show a history of polyploidy – a condition that offers the evolutionary advantage of multiple gene copies yet poses challenges to basic cellular processes, gene expression, and epigenetic regulation. To date, understanding the genomic consequences of polyploidy has been limited by the sparse sampling of groups of taxa with multiple polyploidy events. Here, we present 33 chromosome-scale genome assemblies from 27 species, including all diploid teosinte species and subspecies. These genomes capture 15 independent polyploid formation events, including the shared whole genome duplication between Zea and sister genus Tripsacum. In maize, the after-effects of polyploidy have been widely studied, showing reduced chromosome number, transposable element (TE) expansions, and biased fractionation of duplicate genes. While we observe these patterns within the genus Zea, 12 other polyploidy events deviate significantly. Those tetraploids and hexaploids retain 40 or 60 chromosomes, have only stochastic TE amplifications, and maintain nearly complete complements of duplicate genes. We hypothesize this lack of genomic response to polyploidy arises from differences in the evolutionary paths to re-establishing diploid genetic inheritance. In most Andropogoneae taxa, polyploidy provides multiple copies that buffer genetic load, whereas in maize and other paleopolyploid taxa, reduced chromosome complements and selective retention of duplicate genes generate novel adaptive combinations. In total, these genomes provide a powerful backdrop for maize geneticists to better understand maize diversity and the evolutionary context of maize genes and alleles.

P18: Convergent genome- and gene-level constraints shape repeated environmental adaptation in grasses

Evolution and Population Genetics Sheng-Kai Hsu (Postdoc)

Hsu, Sheng-Kai1
Schulz, Aimee J2
Hale, Charles O2
Costa-Neto, Germano1
Miller, Zachary R1
Stitzer, Michelle C1
Wrightman, Travis2
Zhai, Jingjing1
Brindisi, Lara1
Oren, Elad1
Luo, Yun1
Konadu, Beatrice2
Ojeda-Rivera, Jonathan O1
AuBuchon-Elder, Taylor3
Kellogg, Elizabeth A3 4
Romay, M Cinta1
Buckler, Edward S1 2 5

1Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA 14853
2Section of Plant Breeding and Genetics, Cornell University, Ithaca, NY USA 14853
3Donald Danforth Plant Science Center, St. Louis, MO, USA 63132
4Arnold Arboretum of Harvard University, Boston, MA, USA 02130
5USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA 14853

Grasses (Poaceae) dominate terrestrial ecosystems and sustain global food security, yet the genomic principles enabling their repeated adaptation to extreme environments remain unresolved. Here, we combine dense phylogenomic sampling, global environmental data, and state-of-the-art nucleotide and protein foundation models to characterize the mutational targets underlying environmental adaptation in grasses. Analyzing 707 genomes from 569 species spanning 17 climate zones, we identify dozens of phylogenetically independent transitions into extreme temperature, water, and soil environments. These repeated adaptations are accompanied by convergent shifts in genome-scale molecular properties, including Nitrogen-to-Carbon balance and biosynthetic energetic cost of the proteome, revealing predictable biochemical constraints imposed by natural selection. At the gene level, through an AI-informed phylogenetic mixed modeling framework, we identified 160-89 conserved genes that repeatedly underlie adaptation to the unique axes of environmental challenges. Together, our results show that grass adaptation is channeled by layered constraints acting at genome-wide, pathway and gene-specific scales, producing predictable evolutionary trajectories across the Poaceae.

P19: Diversity of root system architecture and aquaporin in response to low nitrogen in maize landraces

Evolution and Population Genetics Joséphine Guyot (Graduate Student)

Guyot, Joséphine1 3
Nicolas, Stéphane2
Faivre-Rampant, Particia2
Draye, Xavier3
Chaumont, François1

1UCLouvain, Louvain Institute of Biomolecular Science and Technology, Croix du Sud 4 L7.07.14, 1348 Louvain-la-Neuve, Belgium
2Institut National de Recherche Agronomique et de l'Environnement (INRAE), Biology and Plant Breeding, 12 route 128, 91172, Gif-sur-Yvette, France
3UCLouvain, Earth and Life Institute, Croix du Sud 2 L7.05.03, 1348 Louvain-la-Neuve, Belgium

The project aims to uncover the genetic diversity of root system architecture (RSA) and aquaporin (AQP) haplotypes, as well as their expression pattern, under low-nitrogen (LN) conditions in maize landraces. This project is part of the SusCrop–ERA–NET project “Mining Allelic Diversity of Maize Landraces for Tolerance to Abiotic and Biotic Stresses” (MineLandDiv), which aims to identify maize landraces and alleles favorable for tolerance to stresses. In this context, we performed early-stage phenotyping of the RSA of 270 landraces from the MineLandDiv panel in the aeroponic root phenotyping platform at UCLouvain (RootPhAir, EMPHASIS ESFRI) under LN conditions. This platform enables non-invasive monitoring of root growth and architecture of 990 plants, with a temporal resolution of 2 hours over approximately three weeks. Following image acquisition and semi-automatic processing, we measured seminal root angle and elongation rate, as well as lateral root branching density and angle. The results revealed substantial phenotypic variation among landraces in LN conditions, supporting the hypothesis of underlying genetic diversity. To associate this phenotypic variation with genomic regions, we are currently conducting genome-wide association studies using single-nucleotide polymorphisms (SNPs) array data. However, SNP arrays can introduce ascertainment bias, limiting the detection of novel or rare variants, particularly in genetically diverse landraces. To address this limitation, we complemented this study with haplotype analysis in key genomic regions using targeted genotyping-by-sequencing data. Our focus was on AQP genes and loci involved in RSA. AQP genes were prioritized due to their role in abiotic stress tolerance, including LN availability, and in RSA development. This strategy will improve the resolution of our genetic analyses, facilitate the identification of candidate genes associated with RSA under LN conditions, and provide deeper insights into the genetic diversity of maize landraces for this functionally relevant gene family.

P20: GWAS study of epistatic interactions with semidominant mutants establishes the molecular identities and pathways affected by natural variants

Evolution and Population Genetics Brian Dilkes (Principal Investigator)

Dilkes, Brian1 2
Kaur, Aman1 2
Khangura, Rajdeep3

1Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 USA
2Center for Plant Biology, Purdue University, West Lafayette, Indiana 47907 USA
3Plant and Agroecosystem Sciences, University of Wisconsin-Madison, Madison, WI 53706 USA

Mutant phenotypes are valuable resources for studying genetic mechanisms that regulate plant physiology, growth, and development. They serve as links between genotype and phenotype, allowing the examination of genetic variations and their effects on the phenotype. The pathways affected by natural variants segregating in the species can be studied by mapping modifiers that are epistatically determined by the mutant allele. This is easily achieved with dominant mutants of maize than can be crossed to diverse maize lines to generate an F1 association mapping population (FOAM). Each F1 family segregates 1:1 for wild type and mutant plants, permitting the observation of background effects in F1 hybrid siblings that differed at the mutant locus. GWAS on the F1 families permits the discovery of such epistatic interactions can help expand the pathway controlling the mutant phenotype when the identity of the mutant gene is known. The extensive genetic diversity captured in the FOAM populations allows for high-resolution mapping of genetic modifiers that affect the mutant phenotype. Integrating these data with the effects of genetic variation on gene expression provides insights into molecular mechanisms underlying the altered phenotype. We have employed this approach on five dominant dwarf mutants, ten dominant lesion mutants, six dominant leaf patterning and polarity mutants, and two dominant metabolic mutants. We identified natural variation that affects the severity of the phenotype due to cis-acting expression variation at the wildtype allele in the mutant heterozygotes, alleles at known protein-protein interacting partners, and established epistatic interactors with a subset of mutants. This demonstrates the value of both traditional developmental and biochemical genetics and identifies natural alleles of phenotypic consequence in key developmental regulators by GWAS.

P21: Genetic characterization of United States and Canadian heirloom maize seed bank

Evolution and Population Genetics Jordan Cummings (Graduate Student)

Cummings, Jordan M.1
Draves, Melissa A.2
Washburn, Jacob D.3
Flint-Garcia, Sherry3
Holland, James B.1 4 5
Gage, Joseph L.1 4

1North Carolina State University; Department of Crop and Soil Sciences; Raleigh, NC, USA 27695
2University of Missouri; Division of Plant Science and Technology; Columbia, MO, USA 65211
3USDA-ARS; Plant Genetics Research Unit; Columbia, MO, USA 65211
4NC Plant Sciences Initiative; Raleigh, NC, USA 27606
5USDA-ARS; Plant Science Research Unit; Raleigh, NC, USA 27695

Nearly all maize grown in the United States today are hybrid cultivars, but this has not always been the case. Prior to the first double-cross hybrid release in 1921, maize varieties were maintained through open pollination and locally adapted through selection for individual plant phenotypes. These open-pollinated (heirloom) populations harbor extensive phenotypic and genotypic diversity, yet remain poorly described and largely absent from modern breeding. Heirloom maize has increasing value in specialty markets, among chefs, organic farmers, and small-scale growers, and it may also contain novel alleles that illuminate the adaptive history of maize as it spread across the United States. Although heirloom maize from Mexico, South America, and Europe has been well cataloged, no equivalent characterization exists for U.S. heirlooms. Our phenotypic evaluation of 990 U.S. and Canadian heirloom varieties reveals substantial variation both within and between populations, with clear trends emerging among subgroups. To further resolve this diversity, we are conducting low-coverage (~2×) whole-genome sequencing of 10 individuals per population. Preliminary analyses of nearly 5,000 individuals from ~750 populations have shown genetic structure and relationships through multidimensional scaling and clustering by geographic origin, flowering time, and additional trait relationships. Generation and analysis of the genotypic data is still underway along with transcriptomics from multiple environments. This project will generate a comprehensive, publicly available genomic and phenotypic resource that will support researchers, breeders, and other stakeholders in leveraging temperate heirloom diversity for modern maize improvement.

P22: Genetic diversity and population structure in sweet corn

Evolution and Population Genetics Derya Taşcilar (Principal Investigator)

Taşcılar, Derya1
Öztürk, Sevim Döndü Kara2
Özkan, Hakan2

1ZEA GEN SEEDS, Ozgur Neighborhood 2347 Street B, No: 16/1, Interior Door No: 6, Yuregir, Adana, Türkiye
2University of Çukurova, Faculty of Agriculture, Departmnt of Field Crops, Sarıçam, Adana, Türkiye, 01250

The Shrunken-2 (sh2) gene plays a key role in determining kernel quality traits in sweet corn (Zea mays saccharata Sturt.). However, the narrow genetic base of sweet corn is considered a significant constraint in breeding programs. Therefore, understanding genetic diversity and population structure among sweet corn genotypes is critical. In this study, genetic diversity and population structure were assessed in a panel of 282 sweet corn genotypes using 23,909 genome-wide single-nucleotide polymorphism (SNP) markers.Genetic relationships among genotypes were analyzed using the Neighbor-Joining (NJ) method, while population structure was inferred using the STRUCTURE software. At K = 2, genotypes were divided into two major clusters; however, differentiation among subpopulations was limited. In contrast, analyses at K = 3 and K = 4 provided a more refined representation of the genetic structure and revealed genetically meaningful subgroups. The NJ phylogenetic tree was largely consistent with the STRUCTURE results, with genotypes clustering into distinct branches.Genotypes representing the Iowa Stiff Stalk Synthetic heterotic group (23Sh2N-279, 23Sh2N-280, 23Sh2N-282, and 23Sh2N-284) and the Lancaster heterotic group (23Sh2N-281 and 23Sh2N-283) formed a distinct subcluster with short branch lengths in the phylogenetic tree, indicating partial genetic differentiation from the remaining genotypes. In the STRUCTURE analyses, these six genotypes were not entirely genetically pure but consistently showed a dominant shared ancestry component, being primarily associated with Q1 at K = 3 and Q4 at K = 4. These results suggest that, despite their close phylogenetic relationships, these genotypes exhibit a complex genetic architecture characterized by genome-wide admixture.Overall, the findings demonstrate that SNP markers are effective for revealing genetic diversity and population structure in sweet corn and provide valuable insights for identifying suitable parental lines and assessing relationships among heterotic groups in sweet corn breeding programs.

P23: Genetic legacies of the first ancient South American state in archaeological maize

Evolution and Population Genetics Heather Chamberlain (Graduate Student)

Chamberlain, Heather N.1 2
Paucar, Elisa3
Goldstein, Paul S.4
Seetharam, Arun5
Vallebueno-Estrada, Miguel A.6
Swarts, Kelly L.6
Baitzel, Sarah I.7
Pinhasi, Ron1
Somerville, Andrew D.8
Hufford, Matthew B.2

1Department of Evolutionary Anthroplogy, University of Vienna, Vienna, Austria
2Department of Ecology, Evolution, & Organismal Biology, Iowa State University, Ames, Iowa, USA
3Universidad Nacional San Antonio Abad del Cusco & Centro de investigación Arqueológica y Antropológica, Sama, Tacna, Perú
4Department of Anthropology, University of California San Diego, La Jolla, California, USA
5Rosen Center for Advanced Computing (RCAC), Purdue University, West Layfayette, Indiana, USA
6Science for Life Laboratory, Swedish University of Agricultural Sciences, Umeå, Sweden
7Department of Anthropology, University of Washington St. Louis, St. Louis, Missouri, USA
8World Languages and Cultures, Iowa State University, Ames, Iowa, USA

Moquegua Valley in South Peru has an ancient legacy of maize cultivation dating back long before European contact. When and in what form maize arrived in South Peru is still a mystery. While maize arrived in Peru ~6000 years before present (BP), documented movement of maize within the Andes and admixture with Mexican varieties suggests a complicated evolutionary history. Over thousands of years, the people of the Moquegua Valley gradually increased maize cultivation, with more diverse subsistence farming dominating for much of the region’s history. Trade networks were well developed between the peoples of the Valley and the high-altitude regions of the Andes, including the Tiwanaku state. This research explores the co-evolution of humans and maize by analyzing genetic changes in ancient maize over 1,500 years of human occupation in the Moquegua Valley. By examining highland and lowland maize alleles and tracking traits under selection using population genetics, we assess how the expansion of the Tiwanaku state influenced maize genomics. Using AMS-dated archaeological maize samples, we map the genetic changes in maize from ancient times to better understand the impact of the Tiwanaku colonization on local maize varieties. Comparisons to modern maize help assess the legacy of these processes and put ancient specimens in context. Moreover, broader impacts will empower farmers through documentation and curation of diverse regional germplasm, provide educational opportunities through training of undergraduate and graduate students, and establish a meaningful link between the past and present for future researchers.

P24: Genomic basis of artificial selection revealed in two high-oil maize populations

Evolution and Population Genetics Binghao Zhao (Postdoc)

Xu, Gen1
Zhao, Binghao1
Wang, Min1
Zhang, Xuan1
Du, Xiaoxia1
Fu, Xiuyi1
Feng, Haiying1
Yun, Tao1
Guo, Jianghua1
Sun, Fei1
Fang, Hui1
Yang, Xiaohong1

1State Key Laboratory of Plant Environmental Resilience and National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China

Kernel oil content (KOC) is a key quality trait in maize breeding, yet despite extensive artificial selection for high-oil lines, the genetic basis underlying KOC accumulation remains poorly understood. Here, we dissect the phenotypic and genomic responses to recurrent selection in two high-oil maize populations, LDHO and KYHO, founded from nine and fourteen elite inbreds and subjected to seven and thirteen selection cycles, respectively. We evaluated 1,503 inbred lines—spanning three LDHO cycles (C1, C4, C7) and five KYHO cycles (C0, C3, C6, C9, C13)—for 11 oil-related traits and generated whole-genome sequencing data for each line. Both populations showed a continuous increase in KOC across selection cycles, with no detectable reduction in genetic gain over time. Despite sustained phenotypic improvement, genome-wide diversity remained largely stable across cycles, whereas lines from later cycles accumulated a higher deleterious mutation load. Selection scans identified 1,013 selective sweeps differentiating early and advanced cycles, with favorable alleles in most sweeps initially monior but rising consistently under selection. SNP- and haplotype-based association analyses uncovered hundreds of loci for oil-related traits, explaining an average of ~70% phenotypic variance. Favorable alleles of the KOC associated loci were strongly enriched in advanced-cycle lines, and ~50% of them co-localized with selective sweeps. We further validated three positively selected genes with functional effects on KOC. Together, our results uncover how artificial selection reshapes the maize genome to increase KOC, and provide a genetic roadmap for accelerating future high-oil maize breeding.

P25: Harnessing genetic diversity to enhance cold stress tolerance in maize through introgression and gene expression analysis

Evolution and Population Genetics Zehta Fazler (Graduate Student)

Fazler, Zehta1 2
Pérez-Limón, Sergio3
Tandukar, Nirwan1 2
Pugliese, Maria4
Cejalvo, Reneliza5
Sawers, Ruairidh3
Álvarez, Rubén R2

1Department of Genetics and Genomics, North Carolina State University, NC
2Department of Molecular and Structural Biology, North Carolina State University, NC
3Department of Plant Science, Penn State University, PA
4Department of Electrical and Computer Engineering, North Carolina State University, NC
5Department of Crop and Soil Sciences, North Carolina State University, NC

Climate change poses a significant threat to crop sustainability, with maize yields projected to decline by up to 24% by 2030 (1). At the same time, maize agriculture is a major source of nitrous oxide (N₂O), a potent greenhouse gas. Planting maize earlier can improve yield and reduce emissions, but it increases exposure to cold temperatures, limiting growth (2). A promising solution is to leverage genetic diversity, specifically introgressions from one of maize’s wild ancestors, Zea mays ssp. mexicana, which has shown potential in enhancing cold tolerance (3). I hypothesize that mexicana introgressions in the Mexican highland variety Palomero Toluqueño (PT) contain critical genetic variants that can enhance cold stress tolerance in modern commercial maize. To better understand cold tolerance, we used a genetic framework of 229 recombinant inbred lines (RILs) derived from the temperate inbred line B73 and PT and grew these RILs under cold stress conditions. As part of an interdisciplinary NSF grant, we will develop a high-throughput image classification model to assess the stress response of each RIL. Using RNA-sequencing data from the RILs during cold stress, we will map expression quantitative trait loci (eQTL) to identify regulatory variants responsible for changes in gene expression. This research will advance our understanding of the genetic basis of cold stress tolerance in maize and provide valuable insights for the development of more resilient commercial varieties through existing genetic diversity.

P26: Harnessing the genetic diversity of Italian traditional maize landraces through landscape genomics

Evolution and Population Genetics Lorenzo Stagnati (Research Scientist)

Lezzi, Alessandra1
Stagnati, Lorenzo1 2
Caproni, Leonardo3
Dell'Acqua, Matteo3
Busconi, Matteo1 2
Lanubile, Alessandra1 2
Marocco, Adriano1 2

1Department of Sustainable Crop Production, Università Cattolica Del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy, 29122
2Research Centre for Biodiversity and Ancient DNA, Università Cattolica Del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy, 29122
3Institute of Plant Sciences, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, Pisa, Italy, 56127

Climate change is the greatest challenge to modern agriculture significantly impacting cropping systems through an increased frequency and intensity of extreme environmental events. Maize, a fundamental crop for global food security is particularly vulnerable, highlighting the urgent need to develop resilient varieties.This study aims to identify significant genes for adaptation to environmental conditions in 140 individuals derived from 28 Italian maize landraces using a landscape genomics approach to support the development of resilient maize genotypes. Landraces were genotyped using genotyping-by-sequencing approach in order to characterize the collection’s diversity. Population genetic studies were conducted to investigate the genetic diversity and structure of the collection. Partial redundancy analysis (pRDA) was subsequently employed to analyse the relationship between climate variables and genetic variation of the materials. Among the 12 ancestral populations identified, both well-defined and highly admixed groups were observed. This degree of admixture was reflected in the clustering analysis and principal component analysis (PCA), although clear differentiation of individual populations was still apparent. pRDA revealed that 30% of the genetic variance in the collection was explained together by climate (45%), geography (11%), and genetic structure (31%). Three potential genomic signals of adaptation were identified in relation to the environmental variability across the sampling sites. The results highlight significant intra-landrace variability within the examined germplasm and reveal unique landraces tied to ancestral lineages. Genetic markers strongly correlated with environmental factors were highlighted supporting the possibility to find useful alleles for adaptation in landraces, thereby serving as key sources for breeding programs aimed at improving stress tolerance and yield stability under climate change.

P27: Homologous chromosome interactions reshape chromatin accessibility contribute to hybrid vigor in maize

Evolution and Population Genetics Tao Zhou (Graduate Student)

Zhou, Tao1
Zhang, Hongwei2
Li, Lin1

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2State Key Laboratory of Crop Gene Resources and Breeding, National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

Hybridization brings together parental chromosomes within a single nucleus, resulting in hybrid vigor, a widespread phenomenon in maize. Previous studies have largely focused on protein-coding sequences; however, how chromosome interactions contribute to hybrid vigor remains poorly understood. Here, we integrated population genetics with single-cell ATAC-seq and bulk RNA-seq analyses in both inbred and hybrid lines. We show that inbred genomes exhibit relatively rigid chromatin accessibility landscapes and constrained gene expression patterns, which limit their vigor under different conditions. In contrast, hybrid genomes display enhanced dynamic regulation of chromatin accessibility coupled with flexible gene expression. We identify genomic regions that gain increased capacity for dynamic chromatin accessibility regulation after hybridization in the Mo17×B73 compared to parental states. Analysis of H3K27ac and H3K4me3 Hi-ChIP data from Mo17×B73 hybrids reveals that the regulatory activity of these dynamically enhanced regions depends on physical interactions between parental homologous chromosomes. Using an artificial selection-derived progeny population from Mo17×B73 and functional validation, we demonstrate that these homologous chromosome interactions directly contribute to heterosis. In conclusion, interactions between homologous chromosomes drive dynamic chromatin accessibility, thereby enhancing vigor. Our findings uncover a regulatory mechanism underlying heterosis and provide a mechanistic basis for the widespread occurrence of hybridization in nature.

P28: Identification of locally adapted loci and convergent evolution within Andropogoneae species

Evolution and Population Genetics Gretta Buttelmann (Graduate Student)

Buttelmann, Gretta L.1
AuBuchon-Elder, Taylor2 3
Menon, Mitra4
Mambakkam, Sowmya4
Bukowski, Robert5
Romay, M. Cinta6
Buckler, Edward S.6 7
Kellogg, Elizabeth A.2 3
Ross-Ibarra, Jeffrey4
Hufford, Matthew B.1

1Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA, USA 50011
2Donald Danforth Plant Science Center, St. Louis, MO, USA 63132
3Missouri Botanical Garden, St. Louis, MO, USA 63110
4Department of Evolution and Ecology, University of California, Davis, CA, USA 95616
5Bioinformatics Facility, Institute of Biotechnology, Cornell University, Ithaca, NY, USA 14853
6Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA 14853
7USDA-ARS, Ithaca, NY, USA 14853

Understanding how species have adapted to their environment, especially within the extremes of their environment, is valuable information that informs conservation, breeding efforts, and basic understanding of the repeatability of evolution. By associating genetic variants with environmental conditions experienced within a species’ niche, insight is obtained regarding the loci contributing to local adaptation. Using data generated as part of the Pan-Andropogoneae project, we aim to identify loci with significant association to climate and soil variables in order to better understand local environmental adaptation across Andropogoneae (Poaceae) species. Whole genome sequencing data was produced for populations sampled across the geographic ranges of 10 Andropogoneae species. Because the genomic data for each species is low depth, the environmental association pipeline used in this project accounts for the uncertainty in the data through estimating allele frequencies and population structure directly from genotype likelihoods. Correlation of allele frequencies with environment is measured through Kendall’s Tau-b. Results from individual alleles were then combined within windows consisting of predicated genes and upstream regions to determine loci significantly associated with environmental adaptation. Orthologous loci shared between species will be analyzed to determine if there is evidence of convergent evolution across Andropogoneae species. We present preliminary results that will later be expanded to include all 10 species in the dataset. Conclusions from this study will broadly inform understanding of loci and environmental conditions that drive local adaptation across Andropogoneae.

P29: Landscape of language: The contribution of ethnolinguistics to Mesoamerican maize genetic diversity

Evolution and Population Genetics Forrest Li (Graduate Student)

Li, Forrest1 2
Snodgrass, Samantha J1 3
Mambakkam, Sowmya1
Sparreo, Luke1
Perez, Claudia1
Runcie, Daniel E1 2
Coop, Graham1 3
Ross-Ibarra, Jeffrey1 2 4

1Department of Evolution and Ecology; University of California, Davis; Davis, CA, USA 95616
2Department of Plant Sciences; University of California, Davis; Davis, CA, USA 95616
3Center for Population Biology; University of California, Davis; Davis, CA, USA 95616
4Genome Center; University of California, Davis; Davis, CA, USA 95616

Geographic, environmental, and anthropological factors pattern genetic diversity in domesticated crop species, but quantifying to what extent has been challenging. Using georeferenced passport data, we can now leverage genetic data and landscape variables to better study the historical demographics and future adaptive potential of the CIMMYT maize seed bank. Here, we leverage sparse pooled sequencing from almost 15,000 maize populations from across the Americas to identify variants associated with multimodal climate and soil variables via environmental GWAS methods. We additionally model migration of maize gene flow using spatial population genetics software such as FEEMS, finding that elevational barriers such as the Sierra Madre contribute the most to migration resistance. To study the anthropological contribution to maize genetic diversity, we expanded on previous work showing that linguistic difference can drive differentiation of maize. We mapped Mesoamerican maize to geographic ranges of Mayan, Uto-Aztecan, and Otomanguean language speakers and computed how much linguistic distance drives genetic variance as compared to known geographic factors such as elevation. We find that within these ethnolinguistic regions, genetic variation is driven primarily by geography or admixture to teosinte (Zea mays spp. mexicana), but is also correlated to human linguistic differences. In addition, linguistic GWAS, Fst outlier analyses, and selection scans via SweeD also identify specific genetic loci associated with ethnolinguistic identity, such as putative candidate genes in the Yucatan Peninsula, indicating that broad-scale human-maize co-evolution studies can help inform region-specific analyses. Our work provides not only new applications in the growing field of spatial population genetics, such as how environment and human interactions result in patterns of maize trade and selection, but also help elucidate landscape-level insights into the population structure and local adaptation of traditional maize varieties.

P30: Localized stimulation of meiotic crossovers through the juxtaposition of heterozygous and homozygous regions

Evolution and Population Genetics Piotr Ziolkowski (Principal Investigator)

Mikhailov, Mikhail E.1
Boideau, Franz2
Szymanska-Lejman, Maja2
Botnari, Vasile1
Ziolkowski, Piotr A.2

1Laboratory of Plant Resistance, Institute of Genetics, Physiology and Plant Protection, Moldova State University, Chisinau, Moldova
2Laboratory of Genome Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland

Meiotic crossovers reshuffle DNA between homologous chromosomes, ensuring faithful chromosome segregation and generating the genetic variation exploited in plant breeding. In Arabidopsis thaliana, it was recently shown that heterozygous segments embedded in an otherwise homozygous genome attract more crossovers than the same intervals in fully heterozygous F₁ hybrids. So far, this phenomenon has only been described in this predominantly self-fertilizing species, raising the question of whether it represents a more general feature of plant meiosis.Here, I will show that a comparable effect also operates in outcrossing maize. Using designed crosses that create spatially confined interhomolog polymorphism, we quantified recombination in defined genomic intervals in otherwise hybrid backgrounds. We find that local crossover frequency can increase by up to threefold compared with fully hybrid genomes. This stimulation is observed in both male and female meiosis and is strongest when the heterozygous segment fully spans the assayed interval, consistent with a local redistribution of crossovers rather than a genome-wide increase in their number.Because Arabidopsis and maize represent distantly related eudicot and monocot lineages, the shared response to juxtaposed heterozygous and homozygous regions points to a conserved mechanism that modulates where crossovers occur along chromosomes. Finally, I will discuss how this principle can be translated into breeding strategies: by strategically arranging blocks of heterozygosity and homozygosity, it should be possible to locally boost recombination, compact linkage blocks, and accelerate introgression of beneficial alleles from donor lines into elite germplasm. I will present a conceptual breeding scheme and potential practical implementations of this approach for crop improvement.

P31: Maize landraces are not distinct populations or genetic lineages

Evolution and Population Genetics Mackenzie Chun (Undergraduate Student)

Chun, Mackenzie1
Magalang, Paulo1
Fairbanks, Regina1
Ross-Ibarra, Jeffrey1

1Department of Evolution and Ecology, University of California, Davis; Davis, CA 95616 USA

Maize landraces are open-pollinated, locally adapted varieties of Zea mays subsp. mays. Such varieties are often selected based on phenotypic characteristics such as flavor, cob size, and yield. While some landrace names were assigned by local farmers, others were assigned by eugenics-inspired Western researchers. Due to the genetic architecture of traits in maize, multiple allele combinations produce the same phenotype. Thus, we hypothesize that landrace names used to classify racial categories of maize based on “genetic differences” are not actually reflective of the true genetic variation. Using principal component analysis and linear regression, we find no evidence of underlying population structure based on landrace designation and a lack of evidence for a significant correlation between landrace name and underlying genetic variation. Our results suggest that while landrace labels are beneficial for farmers to quickly identify varieties to purchase or trade seed, using landrace labels in a scientific environment under the assumption that underlying genetic variation is being properly represented is erroneous. It is important to acknowledge how eugenics and racism—ideologies that started with humans—also have effects in the scientific research of non-human organisms. In doing so, we also must refuse to remain complacent in its lingering influence on how genetic variation is categorized and conceptualized within the research community beyond the field of human genetics.

P32: Pan-genome and AI identify causal variants for maize highland adaptation

Evolution and Population Genetics Wei-Yun Lai (Postdoc)

Lai, Wei-Yun1
Berthel, Ana1
Casstevens, Terry1
Costa-Neto, Germano1
Franco, Jose A. V.2
Hale, Charlie2
Hsu, Sheng-Kai1
Miller, Zachary R1
Romay, M. Cinta1
Stitzer, Michelle C1
Johnson, Lynn C1
Zhai, Jingjing1
Buckler, Edward S1 2 3

1Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA 14853
2Section of Plant Breeding and Genetics, Cornell University, Ithaca, NY USA 14853
3USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA 14853

Maize (Zea mays ssp. mays) was domesticated in tropical lowlands but later expanded into highland and temperate regions that impose strong cold stress during early development. Although highland landraces show enhanced seedling cold tolerance and accelerated development, the evolutionary origins and molecular basis of these adaptations remain unclear, in part due to marker sparsity and linkage disequilibrium. To overcome these limitations, we generated 17 high-quality long-read maize genomes capturing tropical diversity and combined them with existing assemblies to construct a Practical Haplotype Graph (PHG) of 80 diverse genomes. The choice of Zea mays ssp. huehuetenangensis, rather than B73, as the reference genome for the PHG significantly reduced reference bias during variant calling, yielding a total of ~250M SNPs. We leveraged this PHG to impute genome-wide genotypes for 3,300 locally adapted maize landraces from skim sequencing data. With this, we test two hypotheses: whether altitude-adaptive alleles reflect the evolutionary legacy of ancient whole-genome duplication (WGD) and introgression from highland teosinte, and whether adaptation primarily targets protein-coding variation rather than regulatory change. We observed an elevated proportion, up to 20%, of highland teosinte haplotypes in the imputed highland landraces. Environmental GWAS identified 454 loci associated with altitude adaptation. Contrary to the prevailing views emphasizing regulatory importance, we observed 3-fold stronger enrichment in coding than regulatory regions among these loci. Coding signals were further enriched 13-fold among syntenic duplicate genes, highlighting the role of ancient WGD in preserving protein variants optimized for diverse thermal regimes. Finally, integrating zero-shot scores from a plant DNA foundation model (PlantCAD) prioritized putative causal variants in 63% of the candidate genes, including HPC1, COP1L, SS7, and SUS6. Together, our results clarify the roles of WGD and protein optimization in maize adaptation and provide a scalable framework for identifying climate-resilient alleles.

P33: Quantifying the anthropogenic effects on maize genetic diversity

Evolution and Population Genetics Samantha Snodgrass (Postdoc)

Snodgrass, Samantha J.1
Li, Forrest1 3
Sparreo, Luke5 6
Gerardo Medina, Santiago4
Mambakkam, Sowmya1
Menon, Mitra7
Perez, Claudia1
Moreno, Andrés4
Runcie, Daniel3
Coop, Graham1 2
Ross-Ibarra, Jeffrey1 2

1Dept. of Evolution and Ecology, University of California Davis, Davis, CA, 95620
2Center of Population Biology, University of California Davis, Davis, CA, 95620
3Dept. of Plant Sciences, University of California Davis, Davis, CA, 95620
4CINVESTAV, Mexico
5Center for Biodiversity and Evolution, New York Botanical Garden, Bronx, NY
6Department of Biological Sciences, the Graduate Center, The City University of New York, NY
7Colossal Bioscience

When describing genetic diversity, environment and wild relative introgression often capture major axes of variation. Yet, for domesticates that rely upon humans for survival, anthropogenic effects often remain unquantified. Maize genetic diversity is unstructured, weakly correlated with geography, and admixture with a highland wild relative drives the largest axis of variation. Given maize depends completely upon humans for long-term persistence and dispersal along with the deep cultural connections with maize agriculture in the Americas, we hypothesize human variation may capture additional variation beyond environment and wild relative introgression. Using publicly available GBS and passport data from around 2,000 traditional maize varieties and 515 whole genome sequences of humans from the Mexican Biobank, we quantify the anthropogenic effect on maize genetic diversity in the Americas. Pairing maize and human samples, Procrustes correlations between maize and human genetic diversity was equivalent to maize and geography. The limited population structure within maize points to the effective mixing of maize by human trade across large geographic distances. Within the limited population structure, no environmental or anthropogenic parameters capture a large proportion of variation genome-wide. Thus, long-distance trade may have played a large role in creating existing patterns of genetic diversity found in traditional varieties.

P34: The HapMap of Tibet landraces reveals the genetic mechanism of its highland adaptation

Evolution and Population Genetics En Luo (Graduate Student)

Luo, En1
Wu, shenshen1
Wang, Yuebin1
Liu, Yuanfang1
Hu, jiajun1
Meng, Zuqing4
Yan, Jianbing1 2 3
Ross-Ibarra, Jeffrey5
Song, Fengping4
Yang, Ning1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, 430070 Wuhan, Hubei, China
3Yazhouwan National Laboratory, Sanya 572024, China
4Plant Sciences College, Xizang Agricultural and Animal Husbandry University, Linzhi, 860000, China
5Department of Evolution and Ecology, Center for Population Biology, Genome Center, University of California Davis, Davis, CA, USA

This study constructed a high-density variation map of Tibetan maize, revealing that Tibetan maize can be divided into two subpopulations: Group 1, primarily represented by Markam, and Group 2, predominantly consisting of Motuo. Markam was introduced from Yunnan and Sichuan, while Motuo originated from Nepal and Yunnan. Convergent selection genes were identified between Tibetan maize and other high-altitude maize varieties, as well as between cultivated maize and wild maize. During the adaptation of maize to the high-altitude, low-temperature environment of Tibet, flowering-time genes underwent positive selection. Gene introgression from Zea mays ssp. mexicana played a regulatory role in the high-altitude adaptation of maize. This research uncovered a set of candidate genes associated with high-altitude adaptation, along with another group of candidate genes related to environmental response

P35: The MexMAGIC population explores the genetic architecture of clinal variation in Mexican native maize

Evolution and Population Genetics Ruairidh Sawers (Principal Investigator)

Sawers, Ruairidh J H1
Perez-Limón, Sergio1
Alonso-Nieves, Ana Laura2
Salazar-Vidal, M. Nancy3
Carcaño-Macías, Jessica4
Gillmor, C. Stewart4
Rellán-Álvarez, Rubén5

1Department of Plant Science, The Pennsylvania State University, University Park, PA 16802, USA
2Departamento de Fitomejoramiento, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro 1923, Colonia Buenavista, Saltillo, Coahuila, 25315, México
3Division of Plant Science and Technology, University of Missouri, Columbia, Missouri 65211, USA
4Unidad de Genómica Avanzada, Cinvestav, Irapuato, Guanajuato 36824, México
5Department of Molecular and Structural Biochemistry, N.C. Plant Sciences Initiative, North Carolina State University, Raleigh, NC 27695

Understanding how organisms adapt to different environments is fundamental to evolutionary biology and crop improvement. Theory predicts that when selective pressures track differences in the environment, a phenotypic cline will be established. However, gradual monotonic change in phenotype does not necessarily reflect similar behavior in adaptive genetic variants. Indeed, when genetic architecture is complex, the interaction of a large number of small-effect loci can generate many functionally equivalent allele combinations with similar adaptive outcomes. To empirically characterize the genetic architecture of local adaptation, we have developed a Multiparent Advanced Generation Intercross population (MexMAGIC) using eight Mexican native maize varieties sourced from distinct agroecological environments. We have mapped two putatively adaptive traits (tassel branching and flowering time) in a mid-elevation common garden trial in Mexico. The genetic architecture of tassel branch number was dominated by a single large effect locus containing the candidate gene ramosa1 (ra1). In contrast, we mapped 11 moderate effect QTL for flowering. Allele effects at ra1 aligned well with a previously reported negative elevational cline in tassel branching, the two alleles sourced from the highlands of central Mexico showing strong negative effects. On the other hand, effects at flowering QTL were not clearly correlated with any one source environmental factor, with different loci contributing to apparently distinct genetic architectures between accelerated flowering in the highlands and precocity in lowland early-maturing varieties.Broadly used genotype-environment association (GEA) approaches identify adaptive loci by direct correlation of allele frequency and source environment, without the need for costly and logistically challenging phenotypic evaluation. GEA rests on the assumption that clinal phenotypic variation will be reflected in corresponding clear patterns in the distribution of adaptive alleles. Our observations in the MexMAGIC suggest that the validity of this assumption will depend on the genetic architecture underpinning local adaptation.

P36: The pangenome graph construction of Zea genus

Evolution and Population Genetics Zheng Luo (Graduate Student)

Luo, Zheng1
Zhu, Yongli1
Wu, Shenshen1
Du, ZeZhen2 3
Xie, Min4
Gui, Songtao1
Wei, Wenjie1
Jia, Anqiang7
Wu, Daochuan1
Zhai, Zhaowei1
Luo, En1
Cao, Yu1
Xiong, Tong1
Yang, Xiaohong8
Yan, Jianbing1 2
Jiao, Wenbiao2 3
Ross-Ibarra, Jeffrey6
Qin, Feng5
Yang, Ning1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, Wuhan 430070, China
3National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China
4BGI-Shenzhen, Shenzhen 518083, China
5State Key Laboratory of Plant Environmental Resilience, College of Biological Science, China Agricultural University, Beijing 100193, China
6Department of Evolution and Ecology, University of California, Davis, CA 95616, USA
7Yazhouwan National Laboratory, Sanya 572024, China
8State Key Laboratory of Plant Physiology and Biochemistry and National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China

Understanding how maize and its wild relatives adapted and diversified requires genomic resources that capture the full range of sequence and structural variations (SVs), variation that is often missed when relying on a single reference genome. To address this gap, we built a genus-level pan-genome for Zea by generating six new, high-quality teosinte assemblies and integrating them with 51 existing teosinte and maize genomes. Using this dataset, we characterized the genomic architecture of Zea. We identified 8,587 core genes, which on average showed longer coding sequences and higher expression levels than dispensable genes. Using thousands of conserved single-copy genes across all species and Tripsacum, we refined the phylogenetic framework of the genus, particularly revising the placement and divergence time of Zea diploperennis, consistent with independent evidence from transposable-element insertion ages. Whole-genome alignments uncovered the extensive structural diversity within Zea, revealing over 11 million SVs, with TE-associated SVs dominating. Lineage-specific expansions of Gypsy and Copia LTR retrotransposons explained much of the genome size variation and divergence across species. To enable population-scale analysis of this complexity, we developed a new SV genotyping method optimized for repeat-rich genomes. Combining a graph-based pan-genome with resequencing data from 507 maize and 240 teosinte accessions, we generated a high-confidence population-level SV map. These SVs captured selective sweeps missed by SNP-based scans and modulated both gene expression and agronomic traits. Finally, we demonstrate additional applications of the pan-genome: ATAC-seq peak calling on the pan-genome identified regulatory elements absent from the reference genome, and SNPs derived from whole-genome alignments improved SNP accuracy by reducing false positives from read-mapping approaches. Overall, this work delivers a comprehensive Zea pan-genome, provides new tools for accurate SV discovery and genotyping, and highlights the critical roles of SVs in maize evolution and improvement.

P38: Updates on silencing of the Ga1 reproductive barrier

Evolution and Population Genetics Elli Cryan (Graduate student now, postdoc when the meeting will occur)

Cryan, Elli1 2 3
Kliebenstein, Daniel J1 3
Ross-Ibarra, Jeffrey2 3

1Department of Plant Sciences; UC Davis; Davis, California, USA 95616
2Department of Evolution and Ecology; UC Davis; Davis, California, USA 95616
3Center for Population Biology; UC Davis; Davis, California, USA 95616

In maize and related grasses, the gametophytic factor (Ga) loci control whether a plant has a reproductive barrier that can exclude outside pollen from successfully growing down the silk and fertilizing the embryo sac. On a population scale, the reproductive barrier can allow plants with an active Ga locus, like Ga1, to avoid producing hybrid seed when pollinated by plants with an inactive Ga locus, like ga1. Previously, we found that the most common ga1 allele is found in almost all modern varieties of maize. We called this allele ga1-Off, and we found that it was associated with production of 24-nt siRNAs that match the sequence of the active Ga1 allele. We further found that many maize lines with the Ga1 allele have methylated gene bodies and are unable to produce the Ga1 reproductive barrier. This year, to test whether these methylated Ga1 alleles might be reactivated in the absence of a functional 24-nt siRNA-directed DNA methylation pathway, we grew heterozygous methylated Ga1/ga1-Off plants in control and Mop1/mop1 mutant backgrounds. Mediator of Paramutation1 (mop1), also known as RNA-Dependent RNA polymerase2 (rdr2), plays an important role in directing generation of 24-nt siRNAs that direct DNA methylation. We measured barrier strength in both control plants and test plants. Surprisingly, we find that both control and test plants display a variety of barrier strengths. In contrast, parental inbred lines uniformly show no barrier. This suggests that reactivation of the barrier might occur whether or not the Mop1 phenotype is fully active. This could imply that weak barriers might occur “spontaneously” when the Ga1 locus is unmethylated, and that it takes more than one generation for the ga1-Off allele to fully silence an active Ga1 barrier.

P39: We found a hole in the phenological trait space of maize: is it a new territory for future adaptation?

Evolution and Population Genetics Randall Wisser (Principal Investigator)

Drouault, Justine1
Wisser, Randall J.1 2

1LEPSE-INRAE, Institut Agro, University of Montpellier, 34060 Montpellier, France
2Department of Plant & Soil Sciences, University of Delaware, Newark, DE 19716

The timing of flowering is central to how plants are adapted across different environments. To study this variation in maize, we measured how temperature and photoperiod affect development across genotypes from tropical and temperate breeding pools. Examining the trait space for these sensitivities revealed a clear separation between tropical and temperate maize. Notably, we found an ‘empty space’ in this map: essentially no genotypes combine early flowering per se with high photoperiod sensitivity, although this combination would theoretically be adaptive. Coupled with findings from experimental evolution studies in maize, we hypothesize that this is a byproduct of the preadaptation history and subsequent breeding of maize, and not indicative of an intrinsic coupling between different pathways of gene regulatory networks or physiological controls of flowering time. Now we question if this represents a new modality for maize flowering time adaptation.

Biochemical and Molecular Genetics

P40: A DUF1230 gene confers quantitative resistance to southern leaf blight and gray leaf spot in maize

Biochemical and Molecular Genetics Ruiyu Zhang (Graduate Student)

Zhang, Ruiyu1 2 3
Wang, Zhe1 2 3
Yang, Qin1 2 3

1College of Agronomy, Northwest A&F University; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100
2State Key Laboratory of Crop Stress Biology for Arid Areas; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100
3Key Laboratory of Maize Biology and Genetic Breeding in Arid Area of Northwest Region, Ministry of Agriculture; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100

Southern leaf blight (SLB) and gray leaf spot (GLS) are two major foliar diseases in maize that cause significant yield losses. Identifying genes conferring multiple disease resistance (MDR) is essential for crop improvement. This study aimed to investigate the role of ZmDUF1230 in disease resistance and its potential molecular mechanism. A previously identified quantitative trait locus (QTL) for SLB resistance in bin 8.03 was analyzed using candidate region-based association analysis in a 282-line maize association panel. Functional validation of the candidate gene, ZmDUF1230, was performed using ethyl methanesulfonate (EMS) mutants and CRISPR-Cas9 knock-out and over-expression lines to assess its role in disease resistance. Subcellular localization was determined, and large-scale transcriptome co-expression analysis, yeast two-hybrid screening, and GST pull-down assays were used to identify and validate ZmDUF1230-interacting proteins. ZmDUF1230 was identified as the most significant candidate gene in the QTL region and was found to encode a conserved Domain of unknown function 1230 (DUF1230) protein. Functional analysis revealed that knock-out and EMS mutant lines exhibited enhanced resistance to both SLB and GLS, while the over-expression lines were more susceptible. ZmDUF1230 was localized in the nucleus, cytoplasm, and chloroplast. Yeast-two-Hybrid screening identified FTSH5, a chloroplast-localized metal peptidase, as a ZmDUF1230-interacting protein. GST pull-down assays confirmed their interaction, and mutation of FTSH5 resulted in decreased SLB resistance. These findings suggest that ZmDUF1230 may negatively regulates disease resistance by modulating the function of FTSH5. This study provides new insights into maize disease resistance mechanisms and potential targets for crop improvement.

P41: A genome-scale model tracks how arbuscular mycorrhizal symbiosis mitigates maize N deficiency stress.

Biochemical and Molecular Genetics Alia Dellagi (Principal Investigator)

Decouard, Bérengère1
Chowdhury, Niaz Bahar2
Saou, Aurélien2
Ampimah, Nicholas2
Rigault, Martine1
Yassine, Mohamad1
Quilleré, Isabelle1
Marmagne, Anne1
Paysant le Roux, Christine4
Launay-Avon, Alexandra4
Guérard, Florence4
Mauve, Caroline4
Gakière, Bertrand4
Lévy-Leduc, Céline3
Barbillon, Pierre3
Courty, Pierre-Emmanuel5
Wipf, Daniel5
Magne, Kévin1
Alunni, Benoît1
Hirel, Bertrand1
Saha, Rajib2
Dellagi, Alia1

1Université Paris-Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant Sciences (IJPB), 78000 Versailles, France
2Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, United States of America
3Université Paris-Saclay, AgroParisTech, INRAE, UMR MIA Paris-Saclay, 91120 Palaiseau, France.
4Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif sur Yvette, France. Université Paris Cité, CNRS, INRAE, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif sur Yvette, France.
5Agroécologie, Institut Agro Dijon, Université de Bourgogne, Institut National de Recherche pour l'Agriculture, l'Alimentation, et l'Environnement (INRAE), Dijon, France.

Leveraging Arbuscular Mycorrhizal (AM) symbiosis in agriculture holds significant potential to reduce nitrogen fertilizer use and mitigate its environmental impact. To fully capitalize on this symbiosis, a deeper understanding of the mechanisms by which Arbuscular Mycorrhizal Fungi (AMF) improve nitrogen metabolism and uptake is essential. In this study, we used the AMF species Rhizophagus irregularis and the cereal crop Zea mays (maize) to investigate transcriptomic and metabolomic changes occurring during AM symbiosis under varying nitrogen levels. Our findings revealed that under low nitrogen conditions, inoculation with Rhizophagus irregularis allowed maize to maintain yield with five times less nitrogen input. By integrating maize transcriptomic data into the genome-scale metabolic model iZMA6517, we accurately predicted maize growth and yield, and identified key metabolic shifts. Our study supports the idea that the pyrimidine metabolic pathway is a crucial component of maize nitrogen metabolism during AM symbiosis under nitrogen-limited conditions. We provide an integrative, multi-omics and mathematical metabolic modelling analysis to give a comprehensive overview of the key metabolic pathways in maize required during symbiosis under limiting N. Candidate fungal genes involved in N metabolism were identified owing to an integration of plant and fungal omics data. This will facilitate the development of more effective strategies for exploiting arbuscular mycorrhizal symbiosis.

P42: A single nucleotide polymorphism in HLT1 enhances maize high-light tolerance and adaptation to high altitudes

Biochemical and Molecular Genetics Lishuan Wu (Postdoc)

Wu, Lishuan1
Li, Jianing1
Tian, Feng1

1State Key Laboratory of Plant Environmental Resilience, National Maize Improvement Center, China Agricultural University, Beijing,100193 China

The conversion of light energy to chemical energy is crucial for plant photosynthesis. However, excess light induces photoinhibition, which impairs photosynthetic efficiency and even reduces crop yield. High-light stress poses a significant challenge to maize cultivation, especially at midday or in high-altitude regions. In maize, the master regulators underlying high-light tolerance remain poorly understood. Here, we identified a single nucleotide polymorphism (SNP) in the CCT transcription factor High-Light Tolerance1 (HLT1), which significantly enhances maize tolerance to high light and promotes adaptation to high-altitude environments. Furthermore, we demonstrated that HLT1 directly represses the expression of the photoprotective gene ZmPsbS. The high-light-tolerant allele enhances photoprotective capacity by attenuating this repression on downstream ZmPsbS. Our findings provide novel insights into the genetic basis of maize environmental adaptation and offer a valuable genetic marker for molecular breeding.

P43: Application of a ternary Agrobacterium-mediated transformation system coupled with morphogenesis regulators enables efficient genome editing in temperate and tropical maize

Biochemical and Molecular Genetics Sophia Gerasimova (Principal Investigator)

Gerasimova, Sophia V.1 2
Pessoa, Mateus A.2
Baiochi Riboldi, Lucas2
Siqueira Pinto, Maísa2
Nonato, Juliana V. A.2
Hernandes Lopes, José2
Bruno, Maria H. F.2
Brant Monteiro, Patrícia2
Kumlehn, Jochen4
Arruda, Paulo2
Yassitepe, Juliana E.C.T.2 3
Dante, Ricardo A.2 3

1Martin Luther University Halle-Wittenberg, Biozentrum; Halle (Saale), Germany 06120
2Genomics for Climate Change Research Center, Unicamp; Campinas, SP, Brazil 13083-875
3Embrapa Agricultura Digital; Campinas, SP, Brazil 13083-886
4Leibniz Institute of Plant Genetics and Crop Plant Research (IPK); Gatersleben, Germany 06466

Genome editing of tropical maize is limited by strong genotype dependence and low transformation efficiency, restricting its application in breeding programs. Many elite tropical lines remain recalcitrant to conventional Agrobacterium-mediated transformation methods. Here, we evaluated a ternary Agrobacterium-mediated transformation system coupled with morphogenic regulators as a platform for efficient multiplex genome editing in temperate and tropical maize genotypes. Genome editing constructs were delivered comprising a conventional binary vector including a plant selectable marker and a ternary vector carrying morphogenic regulator genes to enhance regeneration competence. Independent transformation experiments were performed across several maize genotypes, including B104 and three tropical inbred lines (CML360, CML444, and CML488). Multiple guide RNAs enabling single and multiplex genome editing were employed as test cases to assess editing efficiency, phenotypic outcomes, and plant recovery. Regenerated plants were screened by PCR to detect transgene presence and by Sanger sequencing to identify target-specific mutations. The ternary system consistently enabled regeneration of edited plants in genotypes that showed limited or no recovery using selection-based binary transformation. Both transgenic and edited but cas9-free plants were obtained. Edited plants exhibited a range of phenotypes consistent with the intended modifications. Importantly, a substantial proportion of plants generated via the ternary system survived to reproductive stages and produced progeny. Phenotypic evaluation of the M2 generation confirmed stable inheritance of edited traits in the absence of detectable transgene integration. These results demonstrate that the combination of a ternary Agrobacterium system with morphogenic regulators provides a robust and efficient platform for genome editing in temperate and tropical maize. This approach significantly increases efficiency, reduces genotype dependency, facilitates recovery of non-transgenic edited plants, and represents a practical and scalable strategy for trait improvement in tropical maize breeding.

P44: Booster: Boosting drought tolerance in key cereals in the era of climate change

Biochemical and Molecular Genetics Vincenzo Rossi (Principal Investigator)

Lanzanova, Chiara1
Banovic Deri, Bojana1
Rossi, Vincenzo1

1Council for Agricultural Research and Economics (CREA), Research Center of Cereal and Industrial Crops, Bergamo, Italy

Prolonged drought due to climate change has a severe impact on agriculture, requiring measures to secure yield stability under water-shortage conditions. This project funded under the EU Horizon Europe program (https://boosterproject.eu/) aims to be a BOOSTER for developing innovative and sustainable strategies to create climate resilient and drought tolerant cereals. Two synergistic strategies will be implemented to achieve this goal. Firstly, a new approach will identify genomic variants in regulatory regions functionally associated with drought tolerance. Novel regulatory elements underlying resilience will inform efficient breeding efforts to create new drought tolerant cereal varieties. Secondly, novel seaweed extracts and microbial biostimulants will be developed as an eco-friendly approach for improving drought resilience. The two strategies will be tested in two cereals with different responsiveness to drought: European maize and Ethiopian teff, a cereal with high genetic similarity to the desiccation tolerant Eragrostis nindensis. BOOSTER will improve drought tolerance in both maize and teff, while simultaneously exploring the potential for transferring species-specific drought responsive features. By exploiting natural genetic variation to achieve drought tolerant genotypes and by developing biostimulants derived from living organisms, BOOSTER will take advantage of the already available natural resources to steer our agriculture towards novel drought tolerant varieties. Importantly, BOOSTER approaches and results are transferable to other crops. A tailored communication/dissemination strategy and a stakeholders’ engagement plan will ensure the expected outcomes and impacts. The project will produce increased maize- and teff-derived biomass resources under harsh drought conditions, will lower irrigation requirement, will strengthen competitiveness of European and African agri-food industry, and will provide concrete examples for improving public awareness about a sustainable use of bio-based technologies. This poster, presented by the project coordinator, along with colleagues from his Italian Institute, illustrates the structure and the strategies of BOOSTER, with also the aim to attract potential future collaborations and use of BOOSTER derived knowledge and products.

P45: Booster: Engineering regulatory regions to improve drought tolerance in maize

Biochemical and Molecular Genetics Yasmine Vanhevel (Postdoc)

Vanhevel, Yasmine1
Engelhorn, Julia2 3
Hartwig, Thomas2
Nelissen, Hilde1

1VIB-UGent Center for Plant Systems Biology, Ghent, Belgium
2Heinrich Heine University Düsseldorf, Faculty of Mathematics and Natural Sciences, Düsseldorf, Germany
3DIADE, University of Montpellier, CIRAD, IRD, Montpellier, France

The “BOOSTER project” addresses the global challenge of climate change-induced drought by focusing on enhancing drought tolerance in maize and teff, two cereal crops with significant economic and nutritional importance. The project also investigates Eragrostis nindensis, a relative of teff and desiccation-tolerant grass which survives extreme and prolonged drought. The project aims to unravel the genetic and molecular mechanisms underlying drought tolerance in these species by identifying functional genetic variants linked to drought tolerance. By understanding these mechanisms, innovative and sustainable varieties can be developed to boost crop resilience. Quantitative allele-specific MOA/RNA-seq analysis in maize and teff will identify genes and cis-regulatory elements (CREs) by analyzing transcriptome, epigenome and cistrome changes under drought and well-watered conditions. Comparative genomics will be used to identify orthologous genes and CREs across maize, teff and E. nindensis, resulting in a list of shared and unique candidate genes and variations associated with drought response. To functionally validate these regions in maize, we will (1) use CRISPR to delete CRE regions and (2) insert regulatory elements derived from maize, teff and/or E. nindensis. This approach will allow us to validate the maize drought cistrome data and test whether specific CREs or elements from more drought tolerant species can improve more drought sensitive species. In the end, these mutants will be assessed for their drought tolerance and potential phenotypes under controlled drought conditions. The outcomes of this project are expected to improve yield stability and food security under drought conditions while reducing irrigation requirements, thereby boosting the advancement of climate-smart agriculture.

P46: Characterization of two terpene synthases involved in maize volatile production

Biochemical and Molecular Genetics Sarah-Maria Riedl (Graduate Student)

Riedl, Sarah-Maria1
Telleria Marloth, Janik1
Schaff, Claudia1
Degenhardt, Jörg1

1Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany, D-06120

Maize is an excellent model to investigate the biosynthesis of terpenes that are involved in the indirect defense of the plant. Zea mays produces a blend of volatile terpenes after herbivore attack, which are able to attract herbivore enemies like specific parasitic wasps. The volatile blend consists mostly of mono-, sesqui-, and homoterpenes, including the tertiary alcohols linalool, nerolidol, and geranyllinalool. The key enzymes of terpene biosynthesis are terpene synthases (TPS), which convert the prenyl diphosphate precursors geranyl diphosphate (GDP), farnesyl diphosphate (FDP), and geranylgeranyl diphosphate (GGDP) into a highly diverse set of terpene compounds. The maize genome contains approximately 40 putative TPS genes, several of which have been characterized. We cloned two of the remaining uncharacterized TPS genes from the inbred lines B73 and A638 for functional characterization in a bacterial expression system and transient transformation of N. benthamiana. The first terpene synthase, TPS18, had the structural features of a monoterpene synthase and produced only a single monoterpene, the acyclic olefin ocimene in the presence of the GDP substrate. The second TPS lacks the signaling peptide usually associated with mono- and diterpene synthases. The enzyme converts GDP into the tertiary monoterpene alcohol linalool, FDP into the corresponding sesquiterpene alcohol (E)-nerolidol, and likewise GGDP into (E,E)-geranyllinalool. The in planta function of this enzyme is most likely that of a (E)-nerolidol synthase in the cytoplasm, but localization studies are needed for confirmation. This enzyme differs from the previously identified maize terpene synthase TPS2, which has the same activity but contains a signal peptide and is expressed in the chloroplast. Since genetic evidence shows that TPS2 is still responsible for the production of volatile (E)-nerolidol, we would like to understand the role of this newly characterized terpene synthase.

P47: Characterization of the maize-Ustilago maydis interaction in a warming climate

Biochemical and Molecular Genetics Karina van der Linde (Principal Investigator)

Schwarz, Christian G1
Hartmann, Finn1
Zier, Christopher2
van der Linde, Karina1

1University of Regensburg; Regensburg, 93047, Germany
2Bavarian Environment Agency; Hof; 95030 Germany

The biotrophic fungus Ustilago maydis, the causal agent of corn smut disease, occurs worldwide. Upon infection, the fungus induces tumor formation in the plant, leading to reductions in biomass and yield, as well as a degradation in the quality of maize silage. Although model studies predict that global warming will increase pathogen pressure on crops such as maize, the effects of the small temperature shifts expected as a result of climate change and the molecular mechanisms by which temperature influences susceptibility are not well understood. Using the German state of Bavaria as a case study, we analyzed historical climate data and future climate projections to define distinct temperature scenarios. These scenarios formed the basis for experiments on maize–U. maydis infection. We recorded phenotypic traits, such as tumor formation, leaf length, and the timing of spore production. Nine of the 18 maize cultivars were examined further by RNA-seq across all temperature conditions, both with and without U. maydis infection. Integrating these transcriptomic and phenotypic datasets enabled us to identify two previously unknown plant factors involved in the maize–U. maydis interaction. Thus, our study provides novel mechanistic insight into how even subtle temperature increases, as expected under climate change, impact corn smut disease. Key findings on the effects of temperature on infection and the latest data on these two novel factors will be presented.

P48: Comparative transcriptomic analysis of low-temperature stress response in maize (Zea mays L.) at VE and V3 growth stages

Biochemical and Molecular Genetics Ana Nikolic (Research Scientist)

Maize is one of the three most essential cereal crops globally alongside wheat and rice based on global acreage and consumption. It is also recognized as the leading crop by total production volume. As a thermophilic crop that originates from tropical areas, maize is vulnerable to temperature stress when it falls below 12°C. Unstable weather conditions resulting from climate change, including low temperatures during early developmental stages, consistently create the risk of yield reduction. Consequently, the objective of the study was to enhance the understanding of mechanisms related to low temperature tolerance from a transcriptomic perspective during the initial stages of development. This study contrasts previously published findings regarding the maize transcriptome’s response to low temperatures (10/8°C) during the emergence phase (VE) with its response at the V3 growth stage. The complete transcriptome profiling was performed on the third leaf of maize during the V3 growth stage, employing design and material previously utilized for VE analysis: 6h and 24h of stress, as well as the control (25°/20°C) plants, on two inbred lines (one susceptible (Ls) and one tolerant (Lt) to low temperature stress). The analysis included RNA extraction, cDNA library synthesis and Next Generation Sequencing (NGS). Results of the analysis revealed a set of common genes related to photosynthesis (protection against photoinhibition and antioxidant mechanisms) and response to abiotic stress (heat shock proteins (HSPs) and heat shock transcription factors (HSFs)) and showing significant differential expression at both growth stages examined though with distinct patterns. Understanding these stage-specific genetic “switches” allows breeders to develop varieties that are resilient to low temperature stress throughout the entire early planting season, rather than just at germination. Key words: low-temperature stress, transcriptomics, photosynthesis, abiotic stress genes Acknowledgement: This research was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, contract number 451-03-136/2025-03/200040 (Maize Research Institute “Zemun Polje”)

P49: Evidence for TOR-linked translational reprogramming during proteome rebalancing in opaque2 seeds

Biochemical and Molecular Genetics Huda Ansaf (Graduate Student)

Ansaf, Huda1
Shrestha, Vivek2
Yobi, Abou1
Flint-Garcia, Sherry3
Angelovici, Ruthie1

1Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
2Bayer Crop Sciences, St. Louis, Missouri, United States 63167
3U.S. Department of Agriculture-Agricultural Research Service, Columbia, MO, 65211, USA

Cereal grains underpin global nutrition, yet their protein quality is limited by low levels of essential amino acids (EAAs) within seed storage proteins (SSPs). Although reducing SSPs typically lowers total EAAs, many mutants—including opaque2—maintain overall amino acid composition through proteome rebalancing, a compensatory adjustment that complicates biofortification efforts. To investigate the molecular basis of this phenomenon, we performed comparative developmental proteomics in wild-type maize and the rebalanced opaque2 mutant. We uncovered extensive remodeling of the translational machinery across seed development, including coordinated regulation of multiple TOR complex (TORC) components and interactors. Phosphoproteomic profiling further revealed dynamic modulation of TOR activity, positioning TOR as a key regulatory node linking translation and amino acid homeostasis during seed maturation. To bridge these findings with natural variation, we conducted candidate gene association mapping for protein-bound amino acid (PBAA) traits in a maize diversity panel. Several TORC-related genes were significantly associated with PBAA composition, reinforcing a role for TOR-linked translational processes in proteome rebalancing. Together, our results highlight TORC and translational control as central regulators of rebalancing in maize seeds and nominate a set of candidate genes as actionable targets for breeding, transgenic, or genome-editing strategies to improve seed protein quality.

P50: Exploring the relationship between cell wall genetics and mycorrhizal symbiosis in maize under low nitrogen stress

Biochemical and Molecular Genetics Sylvie Coursol (Research Scientist)

Brandicourt, Titouan1
Da Costa, Anaïs1
Rigault, Martine1
Quilleré, Isabelle1
López-Malvar, Ana1
Lima, Stephen1
Laurens, Sophie2
Bunel, Léo2
Le Ru, Aurélie3
Le Brusq, Michel1
Guillaume, Sophie1
Jacquemot, Marie-Pierre1
Griveau, Yves1
Rogowsky, Peter4
Méchin, Valérie1
Reymond, Matthieu1
Reyt, Guilhem2
Dellagi, Alia1
Coursol, Sylvie1
Horlow, Christine1

1Université Paris-Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant Sciences, 78000, Versailles, France
2LIPME, Université de Toulouse, INRAE, CNRS, Castanet Tolosan 31326, France
3Fédération de Recherche 3450, Plateforme Imagerie, Pôle de Biotechnologie Végétale, Castanet-Tolosan 31320, France
4Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, 69364 Lyon, France

Maize is a cornerstone of the French forage system, where yield and silage feeding value are key criteria for hybrid registration. Silage feeding value is strongly correlated with dry matter digestibility, which largely depends on genetically controlled cell wall properties. Nitrogen availability strongly limits maize productivity and is commonly supplied through synthetic fertilizers, raising environmental and economic concerns. In this context, arbuscular mycorrhizal fungi represent a promising biological strategy to enhance nutrient acquisition under low-nitrogen stress. Root cell wall composition plays a central role in regulating nutrient transport, including nitrogen. We therefore investigated how genetic modifications of cell wall composition influence mycorrhizal colonization, as well as root and shoot cell wall digestibility under low-nitrogen treatment. This study relies on maize mutants affected in lignin biosynthesis or hemicellulose composition, providing a genetic framework to dissect cell wall-symbiosis interactions. We will present genotype-dependent effects of arbuscular mycorrhizal fungi inoculation on root traits, including mycorrhizal symbiosis, cell wall digestibility, lignin content, and lignin distribution. We will then show how these root-level changes extend to the aerial part of the plant, affecting cell wall digestibility and lignin content in the whole plant excluding ears, as well as lignin distribution in internodes, under low-nitrogen treatment.

P51: False positive LBD interactions in yeast one-hybrid screens: A plasmid effect

Biochemical and Molecular Genetics Mark Lubkowitz (Principal Investigator)

Frenkiewich, Ayla1
Payne-Hoover, Dell1
Carr, Spencer1
DeVito, Sean1
Khanal, Barsha1
King, Tyler1
Kristoffersen, Helene1
Manahan, Cole1
McDonough, Sam1
Pelletier, Jillian1
Ruiz, Angelina1
Simonson, Sebastian1
Vaughn, Madelyn1
Scanlon, Michael J2
Agbola, Paul3
Bass, Hank3
Lubkowitz, Mark3

1Saint Michael's College, Colchester, VT, USA 05439
2Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA 14853
3Department of Biological Science, Florida State University, Tallahassee, FL, USA 32306

Yeast one-hybrid (Y1H) assays are widely used to identify transcription factors that bind specific DNA sequences in vivo, but like all screens, run the risk of false positives. We performed Y1H screens using promoters of NARROWSHEATH1, LIGULELESS1, LIGULELESS3, and ROUGHSHEATH1—genes expressed in the pre-ligular band during maize leaf development—to identify regulators of leaf angle. All four promoters were bound by a subset of four Lateral Boundary Domain (LBD) transcription factors, which other maize researchers have reported as well. To identify LBD binding sites, we analyzed 29 promoters that interact with LBDs in Y1H assays but found no common cis-regulatory motif. Surprisingly, most promoters lacked the putative LBD consensus sequence (GCGGCG) or a close derivative. However, we identified three potential LBD bindings sites in the parent plasmid pMW2, suggesting a source of false positives. To test this, we removed these sites from several bait constructs and rescreened them. Edited baits exhibited altered LBD binding, indicating that vector-derived sequences can influence Y1H results. These findings suggest that some LBD interactions detected in Y1H screens in maize may be false positives.

P52: Flavonoid-mediated resistance to lepidopteran pests in maize operates through insect gut damage and microbiome disruption

Biochemical and Molecular Genetics Charles Colvin (Undergraduate Student)

Colvin, Charles F1
Lesko, Tyler K1
Chatterjee, Debamalya J2
Chopra, Surinder1

1Department of Plant Science; The Pennsylvania State University; University Park; Pennsylvania; United States; 16802
2Biology Department; Skidmore College; Saratoga Springs; New York; United States; 12866

Maize (Zea mays) production is constrained by lepidopteran pests such as corn earworm (Helicoverpa zea) and fall armyworm (Spodoptera frugiperda), which cause substantial yield losses and drive reliance on chemical insecticides. As resistance to existing control strategies increases, identifying genetically encoded, sustainable sources of insect resistance is a major goal of crop improvement. Flavonoids are a diverse class of plant secondary metabolites with known roles in plant defense, yet the mechanisms by which they affect insect herbivores remain incompletely understood. Here, we evaluated the effects of high-flavonoid maize lines on CEW and FAW performance and investigated the physiological and microbiome-mediated basis of flavonoid-induced mortality. Feeding assays revealed significantly increased mortality and reduced growth in both CEW and FAW larvae consuming high-flavonoid diets compared to low-flavonoid controls. Histological analyses showed pronounced damage to the larval gut epithelium in both species. Profiling of gut bacterial communities using 16S rRNA amplicon sequencing demonstrated that flavonoid exposure substantially altered microbiome composition, including enrichment of taxa associated with pathogenicity. Notably, axenic (germ-free) FAW larvae did not exhibit increased mortality when fed flavonoid-rich diets, indicating that microbial interactions are required for the insecticidal effect. Consistent with this model, transcriptomic and proteomic analyses of CEW and FAW larvae revealed elevated expression of immune-related genes and proteins, including antimicrobial peptides and markers of gut barrier disruption. Together, these results support a conserved mechanism in which maize flavonoids compromise insect gut integrity, promote microbiome dysbiosis, and trigger immune responses that collectively reduce pest survival. This work highlights the potential of leveraging plant secondary metabolism and plant–insect–microbe interactions to develop pest-resistant maize varieties.

P54: Functional analysis of genes ZmGAR2 controlling maize kernel dehydration rate

Biochemical and Molecular Genetics Longyu Liu (Graduate Student)

Liu, Longyu1
Li, Wenqiang1
Yan, Jianbing1

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University

[Objective] Kernel moisture content dynamics in maize constitute a key trait affecting mechanical harvesting and kernel quality. Accelerating the rate of maize kernel dehydration will not only benefit mechanical kernel yield but also improve economic benefits. [Method and Result] In this previous study, we located and cloned ZmGAR2, a candidate gene for the major QTL of maize kernel water using genome-wide association analysis. ZmGAR2 was expressed at a high level in the early stage of kernel development. Knocking out the gene of interest slowed down the dehydration rate of corn kernels and made the kernels larger. In addition, ZmGAR2 may affect plant height and bract senescence. We preliminarily speculated that variation in the 5’UTR region of ZmGAR2 affected the expression of the gene and thus the phenotype. The molecular mechanism is still being studied, and we will explore the reasons why genes affect kernel moisture in maize at the physiological level through subcellular localization and screening of interacting proteins. [Conclusion] ZmGAR2 may affect the water content at the maturity stage of kernels by affecting the water content in the early stages of kernel development.

P55: Genetic dissection of WRKY125 and LOX4 reveals lipid–hormone defense networks against Fusarium verticillioides in maize

Biochemical and Molecular Genetics Alessandra Lanubile (Principal Investigator)

Lanubile, Alessandra1
Ottaviani, Letizia1
Lefeuvre, Rozenn2
Montes, Emilie2
Giorni, Paola1
Dall'Asta, Chiara3
Mithöfer, Axel4
Widiez, Thomas2
Marocco, Adriano1

1Department of Sustainable Crop Production, Catholic University of the Sacred Heart, Piacenza 29122, Italy
2Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon F-69342, France
3Department of Food and Drug, University of Parma, Parma 43124, Italy
4Research Group Plant Defense Physiology, Max Planck Institute for Chemical Ecology, Jena D-07745, Germany

Fusarium verticillioides (Fv) is a major cereal pathogen causing stalk rot and ear rot in maize, negatively affecting crop productivity, and compromising food safety by producing the secondary metabolites fumonisins. While genomic studies have identified candidate resistance loci, the molecular networks underlying effective defense remain poorly defined. Here we combine genetics, transcriptomics, and lipidomics to dissect lipid- and hormone-mediated resistance mechanisms involving the transcription factor WRKY125 and the lipoxygenase LOX4. Previous RNA-seq and GWAS analyses highlighted WRKY125 and LOX genes as putative regulators of plant resistance. To functionally validate these candidates, we generated CRISPR/Cas9-editing wrky125 lines and LOX4 overexpression lines and demonstrated their implication in the resistance mechanisms against Fv. Notably, wrky125 mutants showed a ~5-fold reduction of Fusarium ear rot severity and a ~4-fold decrease in fumonisin accumulation compared to the wild-type plants. RNA-seq analysis highlighted an enhanced modulation of the jasmonic acid (JA) and abscisic acid (ABA) hormones, redox state, cell wall modification, and secondary metabolism-associated genes following fungal infection in the mutant background in contrast to wild-type. These transcriptional changes were accompanied by increased accumulation of JA and ABA, indicating a potentiated hormone-dependent defense response in the absence of WRKY125. LOX4 overexpression also significantly enhanced resistance to Fv infection and reduced fumonisin contamination. Transcriptomic and lipidomic analyses revealed that LOX4 mutants up-regulated expression of 9-LOX and JA-related genes, thereby increasing the production of 9-oxylipins and JA-related metabolites under fungal infection. Collectively, our results demonstrate that WRKY125 and LOX4 converge on lipid–hormone defense networks that restrict fungal colonization and mycotoxin accumulation. These findings provide mechanistic insights into maize resistance and highlight WRKY- and LOX-mediated pathways as promising targets for developing Fv-resistant crops.

P56: Genetic diversity study of farmers’ maize accessions from Nigeria revealed by morphological traits to asses environmental impacts and ssr markers

Biochemical and Molecular Genetics Oyenike Adeyemo (Principal Investigator)

Adeyemi, Omotola R.1
Adeyemo, Oyenike A.1
Adedugba, Adeyemi A.1
Odokina, Anita E.1
Elegbede, Aishat O.1
Chima, Grace N.1
Salawu, Oyiza, S.1
Adekile, Oluwademiladeife J.1
Ifechukwu, Favour C.1

11Department of Cell Biology and Genetics, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria,

ABSTRACT Maize (Zea mays L.) is a staple crop, which is cultivated in diverse agro-economic environments. This study assessed the genetic diversity of 25 farmers’ maize accessions, comprising 16 from northern and 9 from southern Nigeria, using 13 morphological traits and 8 SSR markers. In the environmental impact investigations, it was found that only the northern accessions reached maturity during the 2025 rainy season (April-July) in Lagos. Dendrogram analysis revealed that the 16 northern lines grouped into two major groups. Through the correlation analysis, there is a highly significant positive correlation (r=0.86, p

P57: Genetic interaction between GL15 and FDL1 modulates juvenile cuticle deposition and leaf permeability in maize

Biochemical and Molecular Genetics Gabriella Consonni (Principal Investigator)

Castorina, Giulia1 3
Domergue, Frédéric2
Consonni, Gabriella1

1Department of Agricultural and Environmental Sciences (DiSAA), Università degli Studi di Milano, 20133 Milan, Italy
2Université de Bordeaux, CNRS, LBM, UMR 5200, F-33140 Villenave d'Ornon, France
3Present address: CREA, Research Centre for Genomics and Bioinformatics, 29017 Fiorenzuola, Italy

The plant cuticle is a hydrophobic layer produced by the epidermis of primary aerial tissues that serves as the primary barrier between the plant surface and the external environment, whose main function is to limit water loss. This study investigated the roles and interactions between the regulatory genes ZmFDL1 and ZmGL15 in modulating juvenile cuticle deposition and function in maize. Expression and lipid analyses, morphological studies, and permeability assays were performed on single and double mutants. Our results showed an additive effect of ZmFDL1 and ZmGL15 transcription factors on wax abundance and an epistatic effect of gl15-S on fdl1-1 in determining cutin deposition.ZmFDL1 has a key role in controlling juvenile cuticle deposition and preventing water loss, while the main role of ZmGL15 is to maintain a juvenile cuticle. Lack of ZmGL15 activity, as observed in the gl15-S mutant, results in the acquisition of a cuticle characterized by a higher cutin content, with increased ω-hydroxy fatty acids (FAs) as well as polyhydroxy FAs, and a lower wax content, with a decrease in both aldehydes and long chain-alcohols. These changes result in an increased water-holding capacity of the seedlings under drought stress conditions.Furthermore, gl15-S has an epistatic effect on the phenotype of the fdl1-1 mutant. In the double fdl1-1 gl15-S mutant, the absence of ZmGL15 activity mitigates the fdl1-1 morphological abnormalities and rescues the increased fdl1-1 cuticle-mediated leaf permeability.

P58: Genome editing accelerates flowering in tropical maize

Biochemical and Molecular Genetics Michael Muszynski (Principal Investigator)

Lee, Kuensub1 2
Hampson, Ella3
Carrillo, Rina3
Kang, Minjeong1 2 4
Ghenov, Fernanda3
Higa, Lauren5
Du, Zhi-Yan5
Yu, Jianming1 2
Wang, Kan1 2
Muszynski, Michael3

1Department of Agronomy, Iowa State University, Ames, Iowa, USA, 50011
2Crop Bioengineering Center, Iowa State University, Ames, Iowa, USA, 50011
3Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USA, 96822
4Interdepartmental Plant Biology, Iowa State University, Ames, Iowa, USA, 50011
5Department of Molecular Biosciences & Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA, 96822

Tropical maize is a rich source of genetic diversity that could enhance temperate maize breeding programs, but its sensitivity to long-day photoperiods, resulting in delayed flowering, limits its widespread use. To overcome this barrier, the Genome Engineering to Sustain Crop Improvement (GETSCI) project used CRISPR/Cas9 to mutate three flowering repressor genes, ZmCCT9, ZmCCT10, and ZmRAP2.7, in the tropical inbred Tzi8. A single sgRNA targeting the first exon of each target gene was combined with an excision cassette carrying the morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) to enable efficient transgenic plant regeneration. Transgenic plants carrying frameshift edits in each target gene were recovered, and subsequent crosses produced two non-transgenic genotypes: a double-edited zmcct10, zmrap2.7 line and a triple-edited zmcct9, zmcct10, zmrap2.7 line. Multiple flowering traits were measured for the edited genotypes and unedited Tzi8 inbred in short-day (Hawaii) and long-day (Iowa) field conditions. Both edited genotypes flowered significantly earlier than Tzi8 in both environments. Notably, under long-day conditions, flowering of the two edited lines overlapped with that of the temperate inbred B73, whereas Tzi8 did not. Together, these results demonstrate that targeted, multiplex gene editing can reduce photoperiod sensitivity in a tropical inbred, expanding access to previously untapped genetic diversity for temperate maize improvement.

P59: Genome editing of gibberellin biosynthetic genes alters plant architecture in tropical maize inbred lines

Biochemical and Molecular Genetics Ricardo A. Dante (Principal Investigator)

Dante, Ricardo A1 2 3
Gerasimova, Sophia V2 3 4
Hernandes-Lopes, José1 3
Pinto, Maísa S1 3
Nonato, Juliana V A1 3
Monteiro, Patricia B1 3
Vieira, Leticia R1 3
Bruno, Maria H F1 3
Pauwels, Laurens5 6
Yassitepe, Juliana E C T1 2 3
Gerhardt, Isabel R1 2 3

1Embrapa Agricultura Digital, 13083-886, Campinas, SP, Brazil
2Genomics for Climate Change Research Center, Universidade Estadual de Campinas, 13083-875, Campinas, SP, Brazil
3Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, 13083-875, Campinas, SP, Brazil
4Martin Luther Universität Halle-Wittenberg, Biozentrum, 06120, Germany
5Department of Biotechnology, Ghent University, Ghent, 9000, Belgium
6VIB Center for Plant Systems Biology, Ghent, 9052, Belgium

Approximately a third of global maize (Zea mays L.) production occurs in tropical regions, where yields remain substantially lower than in temperate environments. A much sought-after trait in tropical germplasm is optimal height reduction leading to enhanced resistance to high-density planting, lodging and drought. A major constraint to biotechnology deployment in maize is the strong genotype dependency of genetic transformation. Ectopic expression of morphogenic regulators (MRs) has emerged as an efficient strategy to mitigate transformation recalcitrance and expand genome editing (GE) capabilities across diverse genotypes.Here, we report efficient CRISPR/Cas9-mediated GE in elite tropical maize lines using an MR- and Agrobacterium-mediated transformation protocol originally optimized for the temperate inbred B104. As a proof of concept, targeting of VIRESCENT YELLOW-LIKE (VYL) was used to generate a readily scorable phenotype. Targeted mutations were induced in leaf protoplasts of B104 and three tropical lines, including two CIMMYT (CML) lines, regardless of a single nucleotide polymorphism within the gRNA seed region. Three out of five tropical lines tested were amenable to stable transformation, with efficiencies up to 6.6%. Across events, 97% of T0 plants carried indels at the target site, which were inherited in the T1 generation. Leveraging our GE capability of tropical lines to address an agronomically relevant trait, targeting of genes encoding gibberellin 20-oxidases (GA20ox), which are key regulators of plant height, were attained in CML lines. Genotyping of T0 and T1 plants by Sanger sequencing, combined with digital and manual phenotyping, revealed heritable phenotypes associated with ga20ox mutations. Modifications include reduced height, stem width, and leaf length and width, generating a genotype collection that exhibits a range of plant size and architecture variation. These results demonstrate reduced genotype dependency and efficient GE in tropical maize lines and highlight the potential of similar strategies for both fundamental studies and trait-oriented applications.

P60: High-throughput protein-protein interaction screening reveals pervasive function of the whole maize genome

Biochemical and Molecular Genetics Xiaoyang Shang (Graduate Student)

Shang, Xiaoyang1
Zhou, Tao1
Li, Lin1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, Wuhan 430070, China

Abstract:[Objective] Most eukaryotic genomes undergo pervasive transcription, including from intergenic regions, but its functional relevance is barely understood. This study systematically investigates the functional expression potential of intergenic regions (IGRs) using maize as a model organism. [Method] By integrating multi-omics data across the entire life cycle of maize, a high-throughput and highly sensitive protein–protein interaction screening system was developed to conduct an in-depth analysis of genome-wide expression, particularly in IGRs, under different abiotic stress conditions. [Result] The multi-omics data demonstrated that transcription and translation activities occur across nearly the entire maize genome, supported by reliable peptide evidence. This interactome constructed by our devised protein-protein interaction screening system on randomly fragmented genomic regions and artificial DNA sequences demonstrated that expressed intergenic regions can exert functions through protein interactions, even from random genomic sequences. Under four abiotic-stress treatments, pervasive expression shows dynamic responsiveness and adaptation to environmental stressors. Using RACE experiments combined with ribosome profiling (Ribo-seq) signals, nine functional genes originating from IGRs were identified, including five novel small peptide genes. [Conclusion] Through multi-omics analysis covering the full life cycle of maize, this study systematically elucidates the prevalence and functional significance of pervasive transcription and translation, indicating that expression from intergenic regions plays a biologically important and adaptive role in environmental stress responses. Keywords: maize, intergenic regions, pervasive transcription, pervasive translation, pervasive function, protein–protein interaction

P61: Identification and characterization of molecular drivers of nitrogen remobilization in maize during grain filling

Biochemical and Molecular Genetics Alexa Park (Graduate Student)

Park, Alexa N1
Kettler, Cody1
Ojeda-Rivera, Jonathan Odilón2
Romay, Cinta2
Okumoto, Sakiko1
Murray, Seth1

1Texas A&M University; USA; College Station, Texas, 77843
2Cornell University; USA; Ithaca, New York, 14853

The maize grain is the primary nitrogen (N) sink, containing up to 70% of the total plant N at the end of the season. While some amount of grain N is crucial for seed viability, grain-N is eventually removed from the agricultural fields and fed to livestock, the process through which much of the N losses to the environment occur. Our vision for CERCA (Circular Economy that Reimagines Corn Agriculture) is to promote in-field nitrogen recycling, including allocating more N to the vegetative tissue instead of grains to enhance N retention in the field. Previous studies revealed that a few % decrease in grain N content is possible without compromising seed viability. To pinpoint molecular levers of N partitioning, we have generated RNA-seq profiles of CERCA hybrid tissues over the grain filling period. Concurrently, tissue-specific N content was quantified, allowing us to correlate gene expression patterns with remobilization patterns and identify the drivers of N remobilization during the grain filling period. We found that under the wet field conditions of 2024, the majority of N accumulating in the kernel was derived from ongoing N uptake and de novo assimilation. This is further supported by the overall leaf N assimilation activity correlating with peak of grain filling. Transcriptomic analyses revealed that amino acid transporters were enriched in kernel tissues before active grain filling, constant with roles establishing sink capacity, but showed low expression during active grain filling. In contrast, expression of these transporters increased in the source leaves during grain filling, indicating that active N accumulation during grain filling may be source driven. This presentation will discuss the methodologies employed, key findings, and their implications for future research and practical applications in maize agriculture.

P62: Identification and functional analysis of Zm RCC controlling leaf rolling in maize

Biochemical and Molecular Genetics Wenkang Chen (Research Scientist)

Li, Yiheng1
Wang, Haotian1
Wang, Yanhui1
Chu, Liangcui1
Song, Baoxing1
Xue, Jiquan1
Zhu, Wanchao1
Xu, Shutu Xu1
Chen, Wenkang1

1Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest Region, College of Agronomy, Northwest A&F University, Yangling, Shaanxi, China, 712100

Increasing planting density is one of the most important strategies for enhancing maize yield per unit area. Moderate leaf rolling decreases mutual shading of leaves and increases the photosynthesis of the population and hence increases the tolerance for high-density planting. Here we identified a maize abaxially rolled leaf1 (arl1) mutant with extreme abaxially rolled leaves. Bulk segregation analysis mapping in an F2 population derived from a single cross between arl1 and inbred line Zheng58 with normal leaves identified the arl1 locus on chromosome 2. Sequential fine-mapping delimited the arl1 locus to a ~80-kb genomic interval containing only one candidate gene, ZmRCC. Sequence alignment between arl1 and Zheng58 identified an 8-bp insertion in the coding region of ZmRCC, which led to a frame shift causing premature transcription termination in arl1 mutant. ZmRCC encodes a putative regulator of chromosome condensation (RCC) family protein, and is an ortholog of UV RESISTANCE LOCUS 8 (UVR8) in Arabidopsis. Given that UVR8 usually mediates photomorphogenic responses and acclimation to UV-B radiation by regulating the transcription of a series of transcription factors and phosphorylation events, we conducted a yeast two-hybrid (Y2H) screen and identified 133 potential interaction partners. These interacting genes are enriched in photosynthetic electron transport, redox homeostasis, and microtubule cytoskeleton organization pathways. Among the potential interacting proteins, we focused on the gene ZmPIFI, which encodes a post-illumination chlorophyll fluorescence increase protein. We confirmed a direct interaction between ZmRCC and ZmPIFI by Y2H assays as well as split firefly LUC complementation assays. These studies provide a theoretical basis and genetic resources for the improvement of dense-planting varieties.

P63: Identification of Zea mays homologues of AtVIHs

Biochemical and Molecular Genetics Klea Lami (Graduate Student)

Lami, Klea1
Gaugler, Verena1
Marcon, Caroline2
Schaaf, Gabriel1

1. Department of Plant Nutrition, Institute of Crop Science and Resource Conservation, University of Bonn, Bonn 53115, Germany
2Crop Functional Genomics,Institute of Crop Science and Resource Conservation, University of Bonn, 53113 Bonn, Germany

ABSTRACT Inositol pyrophosphates (PP-InsPs) are crucial signaling molecules in eukaryotes, regulating processes such as energy balance and phosphate (Pi) signaling. In Arabidopsis thaliana, the bifunctional diphosphoinositol pentakisphosphate kinases VIH1 and VIH2 synthesize higher-energy PP-InsPs, including InsP8, through an N-terminal kinase activity. Genetic evidence shows that disrupting VIH1/VIH2 activity impairs growth and can trigger constitutive phosphate-starvation responses, consistent with InsP8 acting as a key intracellular phosphate signaling. Mechanistically, InsP8​ promotes SPX–PHR interactions that restrain phosphate-starvation transcriptional programs when phosphate is sufficient.Beyond nutrition, VIH2-dependent InsP8​ is induced by jasmonate and is required for full jasmonate perception and defense against herbivores and necrotrophic fungi, linking PP-InsP metabolism to hormone signaling and immunity. Building on this framework, we aim to identify and functionally characterize VIH homologues in Zea mays to determine whether PP-InsP-driven control of SPX–PHR phosphate signaling and jasmonate-linked stress responses is conserved in a major crop. References: [1] D. Laha et al., VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate Dependent Defenses in Arabidopsis. Plant Cell 27, 1082-1097 (2015). [2] J. Zhu et al., Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis. Elife 8, (2019). [3] E. Riemer et al., ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis. Mol Plant 14, 1864-1880 (2021). [4] N. P. Laha et al., INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-Dependent Inositol Polyphosphates Regulate Auxin Responses in Arabidopsis. Plant Physiol 28, 2722-2738 [5] P. Gaugler et al., Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers. Biochemistry 61, 1213-1227 (2022). [6] D. Y. Qiu et al., Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionizationmass spectrometry. Nature communications 11, (2020).

P64: Identification of transcription factors that bind to MOA-seq motifs using the yeast 1-hybrid (Y1H) assay

Biochemical and Molecular Genetics Paul Agbola (Graduate Student)

Agbola, Paul S.1
Sayad, Becca M.1
Lubkowitz, Mark2
Bass, Hank W.1

1Department of Biological Science, Florida State University, Tallahassee, FL, USA
2Department of Biology, Saint Michael's College, Colchester, VT, USA

We seek to understand how maize regulates growth, development, and stress responses at the level of chromatin structure and genomic response. To fully understand gene regulatory networks, it is necessary to define the complete set of cis-acting elements located near or within a few base pairs of their corresponding genes. These elements are often bound by their cognate trans-acting factors within accessible chromatin regions. The genome-wide collection of occupied cis-acting elements, known as the cistrome, forms the foundation for understanding transcriptional control in maize and, by extension, other model organisms. We developed MNase-defined cistrome occupancy analysis (MOA-seq) to pinpoint the locations of more than 100,000 potential genetic switches within accessible chromatin regions (Savadel et al., 2021, PMID 34383745). Our MOA-seq analysis defined 215 DNA motif families, but the assay does not reveal the protein-binding partners. To address this gap, we have begun to employ a powerful in vivo genetic assay, the yeast one-hybrid (Y1H) system, to define cognate TFs that bind our MOA-seq motifs. For cis-element “baits”, we used the following motifs for initial screening: om126 (wwTATATAww); om124 (ccCATCTCGyc); om125 (ssCCGACCsv); and dym12 (grGrGrGAGAGrGAGAGAGArrrr). These were screened against a maize 2,000 TF library, yielding several hits which will be validated by other DNA-protein binding assays. We also compared the MOA motifs to published DAP-seq motifs (Galli et al., 2025, PMID: 40506505) for known individual maize TFs and found similarities between them, but relatively few exact matches. This research will advance our understanding of the maize cistrome and its cognate TFs. By linking the cistrome to the TF proteome using Y1H as a eukaryotic promoter assay, we can better elucidate the molecular networks that underpin gene regulation that controls plant growth, development, and stress resilience.

P65: Improved soil quality confers bacterial resistance against rainfall perturbations to mitigate maize yield loss

Biochemical and Molecular Genetics Yangbo Huai (Graduate Student)

Huai, Yangbo1 3
Li, Yonghua3
Yang, Lu5
Ning, Peng3 4
Yu, Peng1 2

1Plant Genetics, TUM School of Life Sciences, Technical University of Munich, Freising, Germany
2Emmy Noether Group Root Functional Biology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany
3State Key Laboratory of Nutrient Use and Management, College of Resources and Environmental Sciences, China Agricultural University, Beijing, China
4National Observation and Research Station of Agriculture Green Development (Quzhou, Hebei), China Agricultural University, Beijing, China
5Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetics Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan, China

Although improved soil quality is widely recognized for enhancing crop stress tolerance, the rhizosphere microbial mechanisms through which high-quality soils mitigate the impacts of extreme rainfall during critical maize growth stages remain poorly understood and largely unvalidated under field conditions. We conducted an 11-year maize field experiment comparing chemical fertilizer alone (CF) with chemical fertilizer plus organic manure (CFM). We characterized rhizosphere bacterial and fungal communities immediately before and after an extreme rainfall event around silking, alongside measurements of soil biochemical properties, maize growth, and yield. Compared with CF, CFM increased the soil fertility index by 265% and doubled soil multifunctionality, and both indices were strongly and positively associated with maize yield resistance to heavy rainfall. Under rainfall perturbation, CFM exhibited a 4.2% higher bacterial community mean tolerance breadth (CMTB) and a lower average variation degree of the bacterial community than CF, whereas fungal CMTB showed no consistent relationship with soil fertility or multifunctionality. Consistently, fewer rainfall-responsive taxa were identified in the CFM rhizosphere. Bacterial co-occurrence network complexity decreased in CF but remained stable in CFM from pre- to post-rainfall, while fungal networks were less complex and less responsive across treatments and rainfall events. CFM also harbored more keystone taxa than CF, most of which correlated positively with soil fertility, multifunctionality, and yield resistance. predicted microbial functional traits involved in carbon, nitrogen, and redox metabolism exhibited less changes in response to rainfall perturbations under the CFM treatment, further indicating greater functional resistance than the CF. Overall, our findings highlighted that, under field conditions, improved soil quality and multifunctionality confers bacterial resistance against climate extremes during maize critical growth to mitigate yield losses. Keywords: Soil quality; Ecosystem multifunctionality; Microbial resistance; Crop productivity; Climate change

P66: Maize Carbohydrate Partitioning Defective1 (Cpd1) acts independently of Carbohydrate Partitioning Defective6 (cpd6) in regulating callose deposition in the phloem

Biochemical and Molecular Genetics Zachary Traylor (Graduate Student)

Traylor, Zachary B.1
Baker, R. Frank1
Flint-Garcia, Sherry2
Braun, David M.1

1Division of Biological Sciences; University of Missouri; Columbia, MO, 65211
2USDA-ARS Plant Genetics Research Unit; Columbia, MO, 65211

As part of plant development, carbohydrates must be transported from photosynthetic source tissues, such as leaves, to carbon-demanding sink tissue, such as roots and kernels, through the process of carbohydrate partitioning. This process is genetically controlled, and disruptions to it lead to deficiencies in carbohydrate trafficking and build-up of sugars in source tissues. The Braun lab has characterized a number of maize carbohydrate partitioning defective (cpd) mutants, however most of these studies have been conducted to understand a single mutant’s effect on carbohydrate flux and not on genetic interactions between two mutants. In this research, we undertook an epistasis analysis to investigate whether two different cpd mutants function in the same or separate pathways. The first mutant, Cpd1, exhibits ectopic callose deposition in the phloem sieve elements. The second mutant, cpd6, displays altered phloem anatomy, including ectopic lignification, but does not show the ectopic callose phenotype. To investigate their genetic interaction, we generated families segregating for double mutants and analyzed the growth and leaf vein anatomy phenotypes. Double mutants displayed a more severe growth and leaf chlorosis phenotype compared to Cpd1 and cpd6 single mutants. Moreover, the Cpd1; cpd6 double mutants exhibited the Cpd1 ectopic callose phenotype in the sieve elements, suggesting that Cpd1 acts independently of cpd6. Elucidation of Cpd1 gene function will provide deeper insights into the process of carbohydrate partitioning and the mechanisms that drive the ectopic deposition of callose in the phloem.

P67: Maize Hex4 and Sh1 G-quadruplex (G4) DNAs function in human viral promoter transcription.

Biochemical and Molecular Genetics Bianca Sheridan (Graduate Student)

Sheridan, Bianca K. M.1
Kortyna, Michelle L.1
Harris, Anna J.1
Francis, Ashwanth C.1
Bass, Hank W.1

1Department of Biological Science, Florida State University, Tallahassee, FL, USA

G-quadruplexes (G4s) are non-canonical DNA structures found in regulatory regions of genes in eukaryotic and prokaryotic genomes. G4 structures are found in telomeres and they also function as gene regulatory elements in promoters across domains in the tree of life, including prokaryotes, plants, and animals. These non-B DNA structures have been extensively studied in relation to human cancer, but the functional role of G4s in plants is not well-characterized. The ubiquitous occurrence of G4s at gene promoters suggests a conserved transcriptional regulatory mechanism in eukaryotes. We set out to investigate this hypothesis by conducting a G4 swap experiment to assess whether G4 structures from maize could functionally replace a G4 in the HIV-1 long terminal repeat (LTR) promoter. We developed a dual fluorescent reporter gene vector to assay and quantify the promoter activity of maize G4s in a human cell line transfection system. We found that maize Hex4 and Sh1 G4s functionally substituted for the HIV-1 G4 element, indicating a mechanistic conservation for G4-mediated transcriptional regulation across domains of life. Our novel dual-fluorescence reporter gene assay provides a robust in vivo methodology to quantify the transcriptional potential of individual G4s. Our findings reveal a surprising functional conservation of G4s as core promoter elements across different biological contexts. Beyond the value of this method to characterize G4s, our research opens new avenues for transcriptional research, plant genetic engineering, and even therapeutic strategies for human pathology.

P68: Maize root gene expression, exudation, and the diversity of rhizosphere microorganisms are slightly affected by moderate soil compaction and soil structure

Biochemical and Molecular Genetics Henrike Würsig (Graduate Student)

Würsig, Henrike1
Santangeli, Michael2
Phalempin, Maxime3
Lippold, Eva3
Bouffaud, Marie-Lara1
Reitz, Thomas1 4 5
Oburger, Eva2
Vetterlein, Doris3 6
Tarkka, Mika1 5

1Helmholtz Center for Environmental Research; Department of Ecology of Agroecosystems; Halle, Germany, 06120
2BOKU University; Department of Ecosystems Management, Climate and Biodiversity; Tulln an der Donau, Austria, 3430
3Helmholtz Center for Environmental Research; Department of Soil System Science; Halle, Germany, 06120
4Martin Luther University Halle-Wittenberg; Institute of Agricultural and Nutritional Sciences – Crop Research Unit; Halle, Germany, 06120
5German Center for Integrative Biodiversity Research (iDiv) Halle-Jena Leipzig; Leipzig, Germany, 04103
6Martin Luther University Halle-Wittenberg; Institute of Agricultural and Nutritional Sciences – Soil Science Unit; Halle, Germany, 06120

Soil compaction and unfavorable soil properties are persistent agricultural problems, that limit plants’ access to water, nutrients, and oxygen. However, root and rhizosphere microbiome responses to soil compaction, and the influence of soil structure and texture in this context, remain poorly understood. We conducted two experiments, investigating the effects of moderate soil compaction and soil structure and texture on maize root gene expression, exudation rates and the rhizosphere ACC-deaminase-carrying (acdS+) microbial community. First, Zea mays L. plants were grown in loam at lower (1.26 g cm-3) and higher (1.4 g cm-3) bulk densities, and, second, grown in finely- and coarsely sieved loam (1.4 g cm-3) and sand (1.5 g cm-3) for 22 days. The novel treatment of coarsely sieved loam aimed to mimic the particle size distribution of sand without altering its chemistry. We expected moderate soil compaction to result in differentially expressed genes (DEGs) related to ethylene signaling, exudation, immunity and cell wall structure, increased exudation rates and changes in the diversity of the acdS+-based microbial community. We hypothesized an influence of soil structure and texture, with coarsely sieved loam behaving more similarly to sand than finely sieved loam. Moderate soil compaction triggered DEGs related to cell wall structure, secondary metabolism, and immunity, higher nitrogen exudation rates, and changes in the diversity of the rhizosphere acdS+-based microbial community. Soil texture also affected root gene expression and exudation rates, while the acdS+-based microbial community was influenced by soil structure and texture. Finally, increasing the coarseness did not have strong effects, suggesting that soil texture matters more than soil structure. Our results demonstrate the sensitivity of roots and the rhizosphere microbiome to soil compaction and the influence of soil structure and texture on these dynamics. This provides new avenues toward management strategies in future farming systems.

P69: Molecular and genetic analysis of a conserved Armadillo Repeat Motif-Containing Protein 7 (ARMC7) involved in U12 splicing

Biochemical and Molecular Genetics Dalton Raymond (Graduate Student)

Raymond, Dalton1
Madlambayan, Gerard1 2
Battistuzzi, Fabia1 2 3
Settles, A. Mark4 5
Lal, Shailesh1 2

1Department of Biological Sciences, Oakland University; Rochester, MI 48309
2Department of Bioengineering, Oakland University; Rochester, MI 48309
3Institute of Data Science, Oakland University; Rochester, MI 48309
4Horticultural Sciences Department, and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611
5NASA Ames Research Center, Mountain View, CA 94041

Splicing of introns is essential for the expression and regulation of eukaryotic genes. Most eukaryotes also have a distinct group of rare introns that are spliced by a different minor spliceosome. Known as U12 introns, this group accounts for less than 0.5% of all introns, and their biological significance remains unclear, despite many reports linking defective U12 splicing to developmental defects in plants and animals. We previously reported that a conserved, novel RNA-binding motif protein 48 (RBM48) is essential for U12 splicing (Bae et al., 2019; Siebert et al., 2022). We showed that RBM48 interacts with armadillo repeat motif–containing protein 7 (ARMC7), and that this interaction is conserved between maize and humans. Later, the RBM48–ARMC7 complex was identified as a core part of the catalytically active minor spliceosome, where It promotes efficient splicing of U12 introns. In this report, we describe a putative zmarmc7 mutant identified in a collection of rgh (rough endosperm) mutants. RT-PCR failed to detect a full-length Armc mRNA in the mutant and revealed defective U12 splicing in only seven of 20 tested minor intron-containing genes (MIGs), indicating a milder U12 splicing defect despite sharing a kernel phenotype very similar to that of previously reported U12 splicing mutants rgh3 and rbm48. We also generated a CRISPR/Cas9 knockout of ARMC7 in human cancer cell lines 293 and THP-1, demonstrating that splicing of U12 introns is disrupted in minor intron containing genes (MIGs) analyzed by RNA-seq and RT-qPCR, providing genetic evidence of human ARMC7’s role in U12 splicing. Cell proliferation assays revealed a significant decrease in mutant cell growth compared to wild-type cells. Furthermore, by monitoring splicing in select MIGs with RT-qPCR analysis, we demonstrate that U12 splicing efficiency depends on ARMC7 levels in a dose-dependent manner. We are currently conducting further RNA-seq to study how ARMC7 affects U12 splicing on a transcriptome-wide scale. Our data highlight an essential role for ARMC7 in U12 splicing, growth, and development.

P70: Molecular and genetic basis of maize cold tolerance and high-latitude adaptation

Biochemical and Molecular Genetics Shuhua Yang (Principal Investigator)

Yang, Shuhua1

1State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Maize (Zea mays), originally domesticated in tropical and subtropical regions, is highly sensitive to cold stress, severely restricts its growth, yield and geographical distribution. Despite its significant agronomic importance, the genetic and molecular mechanisms underlying cold adaptation in maize remains elusive. Through integrated research approaches, we have identified several key regulators that regulate maize cold tolerance, including ZmRR1, bZIP68, ZmICE1, TIP4;3 and HSF21. More recently, we identified a bHLH transcription factor Cold-Responsive Operation LoCUS 1 (COOL1) as a crucial regulator of maize cold tolerance through genome-wide association studies. Natural variations in the COOL1 promoter affects the binding affinity of ELONGATED HYPOCOTYL5 (HY5), a transcriptional factor repressing COOL1 transcription. COOL1 in turn negatively regulates downstream cold responsive genes, thereby modulating cold tolerance. Moreover, calcium-dependent protein kinase CPK17 translocates to the nucleus and stabilizes COOL1 in response to cold stress. Intriguingly, the cold-tolerant allele of COOL1 was predominantly distributed in northern high latitudes with cold climate. This study defines a previously unknown pathway, by which the COOL1-centered module regulates cold tolerance and latitudinal adaptation in maize. Together, these findings provide important insights into the molecular basis of maize cold adaptation and offer promising targets for breeding cold-resilient maize varieties.

P71: Nuclei isolation rfficiency across maize inbred lines

Biochemical and Molecular Genetics Abigail McCarthy (Undergraduate Student)

McCarthy, Abigail I1
Rodriguez, Carmen1
Ou, Shujun1

1318 W 12th ave. Columbus, Ohio, United States 43210

Isolation of intact nuclei is a critical first step for molecular assays used in epigenomic and transcriptomic profiling. However, nuclei yield and integrity can vary depending on tissue type, stress conditions, and genetic background. To assess the influence of genetic background, we compared nuclei isolation efficiency across five maize inbred lines (OH43, OH7B, NC350, NC358, and B73) under non-stressed seedling conditions. Nuclei were isolated from ten-day old seedling leaf tissue using a standardized protocol and counted with a hemocytometer, with yield expressed as total nuclei and nuclei per µl. We hypothesize that genetic differences among inbred lines contribute to variation in both nuclei yield and integrity. Replicated counts across lines were analyzed by one-way ANOVA to test for significant differences in yield and percent intact nuclei. These results suggest that some inbreds yield consistently higher nuclei counts than others, indicating that genetic background affects the efficiency of nuclei isolation. Moving forward, these isolated nuclei will be used for Fiber-seq, a novel sequencing method that methylates open chromatin to profile chromatin architecture. We will apply this approach to investigate how transposons contribute to chromatin dynamics and cold stress responses, comparing tropical and temperate maize lineages to uncover lineage-specific regulatory adaptations.

P72: Overexpression of ZmKRN8 elite allele enhances kernel row number and kernel yield in maize

Biochemical and Molecular Genetics Yuanru Wang (Undergraduate Student)

Sun, Qin1 2
Qin, Yao1 2 3
Hu, Aoqing1 2
Wang, Yuanru1 2
Hu, Boyang1 2
Gong, Dianming1 2
Pan, Zhenyuan1 2
Qiu, Fazhan1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China
2Hubei Hongshan Laboratory, Wuhan, China
3State Key Laboratory of Wheat Breeding, College of Agronomy, Shandong Agricultural University, Taian, China

Kernel row number (KRN) is a crucial factor in determining maize yield composition. Therefore, characterizing more causal genes underlying KRN quantitative trait loci (QTL) will provide valuable genetic resources for optimizing maize yield. Here, we show that a START domain-containing protein encoding gene KERNEL ROW NUMBER 8(KRN8) determines ear-inflorescence meristem (IM) diameter, kernel row number and ear diameter. Further a non-synonymous mutation (A493T) in ZmKRN8 was found to significantly increase both KRN and ear diameter. Overexpression of ZmKRN8 enhanced KRN in maize hybrids of different genetic backgrounds and did not alter other yield-related traits including ear length and hundred kernel weight, which ultimately significantly enhanced maize yield per plant. This finding provides knowledge basis to enhance maize hybrids kernel yield.

P73: PEN1 catalyzes RNA primer removal during plastid DNA replication in maize

Biochemical and Molecular Genetics Xing Huang (Postdoc)

Huang, Xing1 6
Shi, Guolong1 2 6
Xiao, Qiao1 6
Feng, Jiaojiao3
Huang, Yongcai4
Wang, Wenqin3 7
Zhang, Yu1 7
Wu, Yongrui1 5 7

1State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology & Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
2University of the Chinese Academy of Sciences, Beijing 100049, China.
3Shanghai Key Laboratory of Plant Molecular Sciences, Shanghai Collaborative Innovation Center of Plant Germplasm Resources Development, College of Life Science, Shanghai Normal University, Shanghai 200233, China.
4State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, China.
5Shanghai Academy of Natural Sciences, Shanghai 200031, China.
6These authors contributed equally.
7Correspondence: yrwu@cemps.ac.cn (Y. W.), yzhang@cemps.ac.cn (Y.Z.), wang2021@shnu.edu.cn (W.W.)

The plastid DNA (ptDNA) replication is initiated by primases, which synthesize RNA primers, following the synthesis of DNA fragments, primers must be removed prior to ligation. However, the enzymes and mechanisms underlying this process are poorly understood. Here, we cloned a gene from maize that encodes a plastid-localized and Mn2+-dependent 5’-3’ exonuclease (designated PEN1) responsible for this process. The pen1 seeds exhibit development and filling defects that intensify across generations. PEN1 cleaves the RNA primers, allowing for the complete excision of ribonucleotides. We reconstituted the plastid RNA primer removal processes in vitro. We also determined the crystal structure of the PEN1-dsDNA binary complex and explained the structural mechanism of the 5′ to 3′ exonuclease activity for the first time. Mutation of Pen1 resulted in the accumulation of ptDNA breaks, thereby compromising plastid function. These studies fill a critical gap that has long existed in the understanding of ptDNA replication.

P74: Post-synthetic cell wall modification initiates defense signaling, enhancing resistance

Biochemical and Molecular Genetics Megan DeTemple (Graduate Student)

DeTemple, Megan1
Bruner, Averie1
Frye, Emily1
Mugisha, Alither1
Swaminathan, Sivakumar1
Zabotina, Olga1

1Iowa State University, Ames, Iowa, 50011

The cell wall is the plant’s first line of defense against microbial pathogens. This complex fabric of polysaccharides is an active barrier against the hundreds of cell wall-degrading enzymes (CWDEs) secreted by pathogens. Recent research suggests that cell wall modifications caused by CWDEs, also known as damage-associated molecular patterns (DAMPs), serve as biological messages that initiate plant defense signaling pathways. However, the details of how CWDEs affect the cell wall structure and the corresponding signal induced by DAMPs are not well understood. Our long-term goal is to understand the exact nature of these cell wall alterations and the downstream plant immune responses. The lab employs a novel approach of expressing individual microbial CWDEs with a secretory peptide in Arabidopsis and, most recently, in Maize. Using this method, the plant cell wall is modified in very specific ways by individual CWDEs, which are normally secreted by pathogens. Using glycome profiling, we demonstrated that the cell wall epitope abundance is changed in our transgenic plants. Using RNA sequencing in addition to RT-qPCR, we revealed that many plant defense genes are significantly upregulated in the transgenic plants compared to wildtype. Interestingly, it appears that different defense genes are upregulated in plants expressing different CWDEs. This immune signaling appears to have significant biological outcomes as both transgenic Arabidopsis and Maize are resistant to Botrytis cinerea and Colletotrichum graminicola fungal infections. Overall, there is evidence to support that the post-synthetic modification of plant cell walls by expressing fungal CWDEs has established a connection between cell wall modifications, immune signaling, and defense response that will be further explored in the future.

P75: Regulation of aquaporin expression by NAC and HSF transcription factors

Biochemical and Molecular Genetics Jean Fontaine (Graduate Student)

Fontaine, Jean H.1
Teirlinckx, Estelle1
Laurent, Maxime1
Yagli, Aliye B.1
Chaumont, François1

1Louvain Institute of Biomolecular Science and Technology, UCLouvain

Aquaporins of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies facilitate water and small solute diffusion across cellular membranes and play key roles in hydraulic regulation, cell expansion, organogenesis and responses to abiotic stress. However, the transcriptional networks that control PIP and TIP expression remain poorly understood. Expression quantitative trait loci (eQTL) mapping performed in our laboratory identified two NAC transcription factors (NAC24, NAC40) and one heat shock factor (HSF13) as candidate regulators of leaf aquaporin genes (PIP1;3, PIP2;5, TIP1;1) (Maistriaux L, Laurent M J. 2024. Plant Physiology 196:368-384). To test these candidates, we combined genomic binding assays, in vitro binding verification, transient transcriptional assays and transgenic approaches. DNA affinity purification sequencing (DAP-seq) revealed high-confidence binding sites and putative target promoters for NAC24 and HSF13; electrophoretic mobility shift assays (EMSA) confirmed direct binding of these TFs to selected PIP/TIP promoters and to enriched motif sequences identified by DAP-seq. Functional promoter activation is being quantified using dual-luciferase transactivation assays in maize mesophyll protoplasts with promoter–reporter constructs co-expressed with the associated TF. To link molecular interactions to physiological outcomes, we generated Arabidopsis thaliana lines constitutively expressing HSF13 and are phenotyping them under controlled growth and abiotic stress regimes (drought, salinity, heat). In parallel, maize lines with HSF13 knockdown/knockout have been obtained and we are generating BMS suspension cells carrying an inducible HSF13 overexpression cassette to permit cell-level assays. Together, these complementary approaches will clarify whether NAC and HSF family members act as general stress-responsive regulators of aquaporins and/or as context-dependent developmental modulators of plant water relations. Elucidating these transcriptional links will advance our understanding of how plants coordinate membrane water transport with environmental and developmental cues.

P76: Root structure- and soil texture-dependent microbial self-organization in the rhizosphere and in specific endorhizosphere cell types of maize

Biochemical and Molecular Genetics Verena Bresgen (Graduate Student)

Bresgen, Verena1
Hochholdinger, Frank1
Yu, Peng2 3

1Crop Functional Genomics, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany
2Plant Genetics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
3Emmy Noether Group Root Functional Biology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany

Plant roots interact with their environment by actively secreting a diverse array of exudates from the endorhizosphere (i.e., all inner root tissues) into the surrounding rhizosphere, which is the narrow soil zone directly influenced by root-derived chemical and biological processes. Microorganisms constitute the central interface between the endorhizosphere and the rhizosphere, mediating nutrient exchange, signaling, and defense. As a consequence, dynamic cascades of feedback loops between roots and soil emerge, shaped by the spatial heterogeneity and structural organization of the root-associated microbiota. Under natural soil conditions, emerging lateral roots and root hairs represent major hotspots for exudate release, thereby selectively recruiting soil-inhabiting microorganisms into the rhizosphere. To functionally dissect the contribution of root developmental traits to microbiome assembly, the maize mutants roothair defective 3 (rth3), impaired in root hair formation, and lateral rootless 1 (lrt1), impaired in lateral root initiation, will be employed. These genetic perturbations will enable us to directly link defects in root architecture to disruptions in microbial community structure within both the endorhizosphere and the rhizosphere. Moreover, two contrasting soil types (loamy sand and silt loam) will be incorporated to assess how edaphic factors modulate root-soil-microbiome interactions across these compartments. The overall goal of this study is to elucidate how soil-grown maize roots and their exudates drive the self-organization, composition and functional differentiation of bacterial and fungal microbiomes in the rhizosphere and endorhizosphere.

P77: Stress-tolerant Bacillus and Paenibacillus from Vellozia spp. as microbial resources for improving maize phosphorus use and drought tolerance

Biochemical and Molecular Genetics Sylvia Morais de Sousa Tinoco (Principal Investigator)

Ferreira, Nataly F.1
Souza, Rafaela F.A.2
Maia, André L.M.2
Gerhardt, Isabel R.3 4
Dante, Ricardo A.3 4
Lana, Ubiraci G.P.5
Tinoco, Sylvia M.S.1 2 3 4 5

1Federal University of São João del-Rei (UFSJ), São João del-Rei, MG, Brazil, 36301-158
2University Center of Sete Lagoas (Unifemm), Sete Lagoas, MG, Brazil, 35701-242
3Genomics for Climate Change Research Center (GCCRC) Campinas, SP, Brazil, 13083-875
4Embrapa Digital Agriculture, Campinas, SP, Brazil, 13083-886
5Embrapa Maize and Sorghum, Sete Lagoas, MG, Brazil, 35701-970

Campo rupestre is a Brazilian ecoregion characterized by very nutrient-poor soils and severe water scarcity. Species of the family Velloziaceae thrive in this environment using different drought-resistance strategies, from desiccation tolerance (Vellozia nivea) to drought avoidance (Vellozia intermedia). The microbiome associated with these plants is a unique and largely unexplored source of stress-adapted microorganisms with potential uses in crop improvement. In this study, we examined Vellozia-associated bacteria to identify plant growth–promoting strains relevant to maize under nutrient and water stress. Endospore-forming bacteria were isolated after heat shock (80 °C), resulting in 128 isolates from 481 initial samples. These isolates were tested for phosphate solubilization using tricalcium phosphate and sodium phytate as inorganic and organic phosphorus sources, respectively, and for tolerance to osmotic and saline stress. Osmotic stress was tested in LB medium with sorbitol (405, 520, and 780 g L⁻¹), and salt tolerance was tested in LB medium with 10% and 20% NaCl (w/v). Among the isolates, 75 solubilized phosphorus from phytate, and 71 from calcium phosphate. Ninety-seven isolates tolerated 405 g L⁻¹ sorbitol, 31 tolerated 520 g L⁻¹, and 12 tolerated 780 g L⁻¹. Salinity tolerance was noted in 88 and 77 isolates at 10% and 20% NaCl, respectively. Eight isolates showed superior performance across all stress tolerance and phosphate solubilization tests. Molecular identification based on 16S rRNA and gyrB gene sequencing mainly revealed Bacillus and Paenibacillus. After genetic screening, 37 strains were tested for plant growth–promoting traits and early maize seedling performance under controlled conditions, with 20 strains further evaluated under PEG-induced osmotic stress. Statistical analyses (p < 0.05) and principal component analysis supported the selection of top strains for greenhouse and field testing. This research highlights stress-tolerant microbial resources that can be integrated into maize genetic and breeding studies to understand genotype × microbiome interactions that influence phosphorus use efficiency and drought tolerance.

P78: Sucrose transporters expressed in the endosperm adjacent to the embryo (EAS) are necessary for carbon partitioning and embryo growth

Biochemical and Molecular Genetics Thomas Widiez (Principal Investigator)

Fierlej, Yannick1
Grazer, Laurine1
Khaled, Abdelsabour G.A.1 2
Langer, Matthias3
Montes, Emilie1
Perez, Thibault4
Gallo, Laura4
Lacombe, Benoît4
Nacry, Philippe4
Doll, Nicolas M.1
Borisjuk, Ljudmilla3
Rolletschek, Hardy3
Ingram, Gwyneth1
Rogowsky, Peter M.1
WIDIEZ, Thomas1

1Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France.
2Department of Genetics, Faculty of Agriculture, Sohag University, Sohag, Egypt.
3Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Seeland Gatersleben, Germany
4IPSiM, Université de Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

In cereals such as maize, the kernel accumulates large quantities of storage compounds, including carbohydrates, lipids, and proteins, and therefore requires tight regulation of nutrient transport for proper seed development. Seeds are composed of distinct tissues: the embryo, the endosperm, and maternal tissues that are symplastically disconnected (not connected through plasmodesmata), necessitating specialized nutrient transfer mechanisms. In maize, nutrient transfer from maternal tissues to the endosperm via specialized basal endosperm transfer layer (BETL) cells is well characterized. However, nutrient transfer at the endosperm/embryo interface remains poorly understood. Consequently, the routes by which maternal carbon–derived sugars support embryo growth are still unclear. Our previous transcriptomic profiling uncovered a novel Endosperm domain Adjacent to the embryo Scutellum (EAS) with strong enrichment for transporter genes. Notably, three sucrose transporters from the SWEET (Sugars Will Eventually be Exported Transporters) family are highly and preferentially expressed in the EAS, suggesting the existence of a specialized sugar transfer mechanism at this interface. We show that these three ZmSWEET proteins are membrane-localized sucrose transporters and are functionally important for kernel development. A gene-edited triple zmsweet14a/14b/15a mutant exhibits reduced embryo size, significantly decreased embryo oil accumulation at maturity, and altered carbon partitioning within the kernel. In addition to developmental defects, mutant kernels display a significant reduction in primary root length during germination, indicating either lasting physiological consequences of disrupted sucrose transport during seed development or an additional role for these SWEET transporters during germination. Together, our findings demonstrate that sucrose transport at the endosperm/embryo interface is critical for proper carbon allocation, embryo development, and seed vigor, and identify the EAS as a key functional domain and potential target for improving seed composition.

P79: Systematic exploration of transcription factor function in maize

Biochemical and Molecular Genetics Taylor Scroggs (Graduate Student)

Scroggs, Taylor1
Nelms, Brad2

1Department of Genetics, University of Georgia, Athens, Ga, USA 30605
2Department of Plant Biology, University of Georgia, Athens, Ga, USA 30605

Transcription factors (TFs) play a wide range of roles in plant growth and development. We have established methods for high-throughput transformation of leaf protoplasts in 384-well plates, and are using this system to test the response of ectopically expressing 2,112 maize TFs. Median transformation efficiencies are greater than 70% and consistent between days and across plates. To date, we have overexpressed 328 individual TFs in duplicate and measured the genes that were induced or repressed by RNA-Seq. Biological replicates show a reproducible response relative to controls for 90% of TFs. On average, there were 102 genes induced and 77 genes repressed per TF. Different TFs generally induced distinct targets, even when they are from the same protein family. In the next steps, we will continue the established workflow through the remainder of the cloned maize TFome, producing a resource for the plant community.

P80: Targeted GBS provides an efficient way to characterize haplotypic diversity and allele frequency in pooled maize landraces.

Biochemical and Molecular Genetics Patricia Faivre Rampant (Research Scientist)

Canaguier, Aurélie1
Minguella, Raphaël2
Madur, Delphine2
Berard, Aurélie1
Le Clainche, Isabelle1
Hinsinger, Damien1
Nicolas, Stéphane2
Faivre Rampant, Patricia1

1Université Paris-Saclay, Centre INRAE Ile de France Versailles-Saclay, EPGV, 2 rue Gaston Crémieux 91000 Evry, France
2Université Paris-Saclay, Centre INRAE Ile de France Versailles-Saclay, IDEEV, GQE, 12 rue 128, 91190 Gif-sur-Yvette, France

Genotyping using pooled DNA represents a cost-effective and powerful strategy to explore genetic variation at the population level. Moreover, DNA pool strategy allows the estimation of allele frequencies from groups of individuals rather than single genotypes, which is particularly relevant for landraces. For more than three decades, single nucleotide polymorphisms (SNPs) have been widely used as molecular markers due to their simple design, genome-wide distribution, and automated detection. However, the biallelic nature of SNPs limits their ability to capture fine-scale genetic diversity, especially in pooled samples. Microhaplotypes, combine neighboring SNPs, providing access to higher levels of polymorphism, well suited for allele frequency estimation in pools. Targeted genotyping-by-sequencing (tGBS) technologies, such as NEBNext Direct Genotyping Solution (New England Biolabs) and Allegro targeted genotyping V2 (Tecan), generate sequence information containing SNPs and should enable microhaplotype reconstruction. We developed a bioinformatics workflow to detect SNPs, construct microhaplotypes, identify alleles, and estimate their frequencies in both individual and pooled samples. Based on previously published maize SNP datasets, two new SNP panels were designed: 110 SNPs for NEBNext and 2,587 SNPs for Allegro, including the former 110 SNPs. tGBS was performed on well-characterized maize material, including B73 replicates, 11 hybrids, 30 controlled pools, their 10 parental inbred lines, and 11 landraces. Statistical analyses showed that while haplotype allele frequency estimates in pools may be biased, allele detection is robust for expected frequencies above 10%, supporting the relevance of microhaplotypes for pooled genotyping. This proof of concept highlights the potential of microhaplotype-based and pool-enabled targeted sequencing approaches for cost-effective genetic diversity characterization in maize and other highly heterozygous plant species.

P81: The JA-induced emission of volatiles in maize is negatively influenced by a bHLH transcription factor

Biochemical and Molecular Genetics Claudia Schaff (Postdoc)

Schaff, Claudia1
Telleria-Marloth, Janik1
Degenhardt, Jörg1

1Institute for Pharmacy, Martin Luther University Halle-Wittenberg, Halle, Sachsen-Anhalt, Germany, 06120

The production of volatile compounds by maize after herbivore attack is mostly mediated by the jasmonate pathway. Recently, the maize analog of Arabidopsis MYC2 was identified which seems to be the master regulator of jasmonic acid production. To understand the regulation mechanisms in response to herbivore attack, we aim to investigate downstream transcription factors leading to the production of volatile terpenes. We identified a transcription factor that is highly upregulated after maize plants were treated with mechanical damage and the application of oral secretion of Spodoptera littoralis larvae. This transcription factor, tf23, shows its highest expression half an hour after treatment and starts to decline after one hour, implicating an early role in the herbivore defense pathway. To further investigate the functional role of this bHLH factor, we generated stable overexpression lines in maize. The overexpression lines of tf23 show a general trend of reduced volatile emission after herbivore induction, with a significant reduction of linalool, geranylacetate, and DMNT ((E)-3,8-dimethyl-1,4,7-nonatriene), indicating a negative role in the regulation of herbivore defense. In accordance with this result, in silico analysis revealed coexpression and putative functional interactions with several jasmonate ZIM domain proteins. In addition, RNA sequencing data of the transgenic maize lines showed a high expression of ZIM14, ZIM16, and ZIM30. On the other hand, we found a significant reduction of expression of a deoxyxylulose phosphate synthase which is responsible for the production of terpenoid precursors. These results indicate that tf23 is a negative regulator in the signaling transduction pathway. Further experiments with a protoplast transformation system will reveal the interactions with the predicted ZIM proteins.

P82: The association of indels with meiotic recombination sites in maize

Biochemical and Molecular Genetics Yike Chen (Undergraduate Student)

Chen, Yike1
Sajai, Nikita1
Mafessoni, Fabrizio2
Pawlowski, Wojciech P1

1School of Integrative Plant Science, Cornell University
2Dipartimento di Scienze della Vita, Universita degli Studi di Trieste

Meiotic recombination is generally considered a high-fidelity process. However, accumulating evidence, largely from human studies, suggests that recombination can be an important source of mutations rather than an error-free repair pathway. We asked whether meiotic recombination in maize could be associated with the creation of indels, which are key contributors to structural variation. Using an indel catalogs derived from the analysis of 26 maize inbred lines generated by the Hufford lab, we quantified indel density around crossover (CO) hotspots as well as hotspots of the formation of meiotic double-strand breaks (DSBs), which initiate meiotic recombination. CO sites showed substantial enrichment relative to random genome sites: 7.65× for indels 1–10 bp in length, 9.38× for indels 100–150 bp; and 2.15× for 0.5–10 kb. DSB hotspots showed only modest enrichment for small (1–10 bp) and medium (100–150 bp) indels and no enrichment of large (0.5–10 kb) indels. Because COs occur in open chromatin, we examined whether chromatin alone could explain indel enrichment. Compared to control sites that are not recombination hotspots but exhibit open chromatin characteristics, CO hotspots still showed excess of small indels, suggesting that chromatin context is not sufficient by itself to account for hotspot-associated indel accumulation and providing evidence that these indels could be formed by meiotic recombination. To probe the underlying repair mechanisms generating these indels, we identified indels that resulted from deletions that took place during the evolution of maize, using Tripsacum as outgroup, and examined indel junctions. Deletions at CO sites showed the most frequent presence of microhomologies at their borders, compared to DSB hotspots and control sites. Together, these results indicate that recombination in maize is a frequent source of indels which primarily arise during CO formation and can be repaired by non-homologous recombination mechanisms.

P83: The characterization of RNA binding motif protein 48 (RBM48) in U12 intron splicing, cellular differentiation, and development

Biochemical and Molecular Genetics Caleb Kienbaum (Undergraduate Student)

Kienbaum, Caleb2
Whitney, Kayla2
Raymond, Dalton1
Madlambayan, Gerard1 2
Battistuzzi, Fabia1 2 3
Lal, Shailesh1 2

1Department of Biological Sciences, Oakland University, Rochester, MI 48309
2Department of Bioengineering, Oakland University, Rochester, MI 48309
3Institute of Data Science, Oakland University, Rochester, MI 48309

Precise removal of introns from pre-mRNA is essential for gene expression in eukaryotes. Most eukaryotic genomes also contain a rare, uniquely structured group of introns spliced by a different minor spliceosome. Known as U12 introns, their biological significance is not fully understood; however, mutations that disrupt U12 intron splicing cause developmental defects in both plants and animals and are linked to several human diseases, including cancer. We previously identified a novel RNA-binding protein, RBM48, which is essential for U12 splicing and conserved between maize and humans (Bae et al., 2019; Siebert et al., 2022). In this report, we demonstrate that this core minor spliceosomal protein produces multiple transcript isoforms ​via alternative splicing. However, the​ physiological role​ of these transcript isoforms in U12 splicing remains unknown. Using RT-PCR expression analysis of several minor intron-containing genes (MIGs), we demonstrate that each transcript isoform, when expressed individually in CRISPR-Cas9-mediated knockout of RBM48 in the human K562 cancer cell line, can complement and restore U12 splicing in the mutant cells. This provides strong genetic evidence of their essential role in U12 splicing. We also show that the efficiency of U12 splicing restoration depends on the level of RBM48 transcript isoform expression in the mutant cell, indicating a dose-dependent effect. Currently, we are conducting RNA-seq analysis to evaluate the impact of each RBM48 transcript isoform and to determine whether they serve redundant or non-redundant roles in U12 intron splicing on a genome-wide scale. The results of these studies will be presented.

P84: The impact of the maize B chromosome on transcriptome during plant development

Biochemical and Molecular Genetics Jan Bartoš (Principal Investigator)

Hloušková, Lucie1
Tulpová, Zuzana1
Holušová, Kateřina1
Karafiatová, Miroslava1
Bartoš, Jan1

1Institute of Experimental Botany of the Czech Academy of Sciences, Šlechtitelů 31, Olomouc, Czech Republic

Maize (Zea mays L.) is a major global crop and an important model organism in biological research. Some maize plants carry supernumerary B chromosomes. Although the maize B chromosome has been well characterised, its transcriptional activity in different plant tissues is not well understood. In this study, we present a comprehensive transcriptomic atlas comparing maize plants with and without the B chromosome across eleven tissues and organs. Our results demonstrate that B chromosome-encoded genes exhibit transcriptional activity throughout plant development, reaching peak expression levels in reproductive tissues. Co-expression analysis revealed a distinct cluster of 30 genes that are specifically expressed in the tassel, suggesting that they are involved in B chromosome-mediated regulation of crossover frequency. In addition to its own transcriptional activity, the B chromosome exerts a widespread influence on the expression of genes located on standard A chromosomes in all examined tissues. Together, our findings provide new insights into the transcriptional landscape of the maize B chromosome and its genome-wide regulatory effects. They also establish a valuable resource for future studies exploring the B chromosome’s broader biological significance.

P85: The maize ZmVPS23L and ZmFBK1 modulate the NLR protein Rp1-D21-mediated defense response by changing its subcellular localization

Biochemical and Molecular Genetics Guan-Feng Wang (Principal Investigator)

Tang, Chang-Xiao1
Sun, Yang1
Wang, Guan-Feng1

1School of Life Sciences, Shandong University, Qingdao, Shandong 266237, China

The interactions between nucleotide-binding, leucine-rich repeat (NLR) proteins and their cognate pathogens often trigger a hypersensitive response (HR). The endosomal sorting complex required for transport (ESCRT) machinery is a conserved multi-subunit complex, which is essential for the biogenesis of the multivesicular body and cargo protein sorting. VPS23 is a key component of ESCRT-I machinery and plays important roles in plant development and abiotic stresses. However, the role of VPS23 in NLR-mediated defense response remains unknown. Here we revealed that ZmVPS23L suppresses the NLR protein Rp1-D21-mediated HR in maize and Nicotiana benthamiana. The variation in the suppressive effect of ZmVPS23L on HR between different ZmVPS23L alleles is correlated with variation in their expression levels. ZmVPS23L is predominantly localized in the endosomes, and it physically interacts with the coiled-coil domain of Rp1-D21 and relocalizes Rp1-D21 from nucleo-cytoplasm to endosomes. We also identified an F-box protein ZmFBK1 modulates the homeostasis of Rp1-D21 and suppresses Rp1-D21-mediated HR. Interestingly, ZmFBK1 is predominantly located in the autophagosome-like dots and it degrades Rp1-D21 by promoting autophagy and interacts with the autophagy-related protein ZmATG6, which also suppresses Rp1-D21-mediated HR. Notably, ZmFBK1 also negatively regulates the resistance to southern corn rust caused by Puccinia polysora and southern leaf blight caused by Cochliobolus heterostrophu, and both pathogens appear to promote ZmFBK1-mediated autophagy in maize. In summary, we demonstrate that ZmVPS23L and ZmFBK1 act as negative regulators in Rp1-D21-mediated HR likely by altering its subcellular localization or causing the protein degradation of Rp1-D21. Our findings reveal a novel role of ZmVPS23L and ZmFBK1 in NLR-mediated defense response. Reference:Chang-Xiao Tang, Shi-Jun Ma, Qi-Dong Ge, Yang Sun, Tong-Tong Liu, Man Yang, Zi-Xuan Kang, Yu-Chen Dai, Xuan Zhang, Xueman Liu, Yanyong Cao and Guan-Feng Wang. 2025. The F-box protein ZmFBK1 facilitates autophagy-mediated degradation of maize NLR protein Rp1-D21 to suppress the hypersensitive response. Molecular Plant, Accepted.Yang Sun, Shijun Ma, Xiangguo Liu, Guan-Feng Wang. 2023. The maize ZmVPS23-like protein relocates the nucleotide-binding leucine-rich repeat protein Rp1-D21 to endosomes and suppresses the defense response. The Plant Cell 35: 2369-2390.

P86: The role of the root microbiome in agronomically important traits of Mexican maize varieties

Biochemical and Molecular Genetics Ana Laura Alonso-Nieves (Principal Investigator)

Alonso-Nieves, Ana Laura1
Rangel-Chávez, Cynthia P2 3
García-Chávez, Jorge N4
Gillmor, Stewart5
Sawers, Ruairidh J H6

1Universidad Autónoma Agraria Antonio Narro, Plant Breeding Department; Buenavista, Saltillo Coahuila, Mexico, 25315.
2Tecnológico Nacional de México/ITS de Irapuato, Division of Biochemical Engineering; El Copal, Irapuato, Guanajuato, México, 36821.
3University of Guanajuato, Department of Food, Life Science Division; El Copal km 9, Irapuato, Guanajuato, México, 36500.
4Universidad Nacional Autónoma de México (UNAM), ENES-León, Laboratory of Agrogenomic Sciences; León, Guanajuato, Mexico, 37684.
5Cinvestav, Unidad de Genómica Avanzada; Irapuato, Guanajuato, México, 36824.
6The Pennsylvania State University, Department of Plant Science; University Park, PA, USA, 16802.

Plant–microorganism associations play a critical role in plant development and performance, especially under unfavorable environmental conditions. Locally cultivated maize varieties have been selected over many generations across diverse environments, resulting in location-specific varieties. These varieties are phenotypically diverse from each other due to genetic differences and the interaction of their environments. For this reason, we aimed to describe the role of the rhizosphere and endosphere microbiome association to agronomically important traits in Mexican maize varieties. We conducted a two-year field experiment to isolate microbiomes associated with ~170 genotypes of Mexican native varieties coming from a multi-parent mapping population. Using ITS1 and 16S rRNA amplicon sequencing, we identify rhizosphere-associated fungi and bacteria exhibiting a significant contribution to kernel length and type, and yield. As previously reported, we found higher diversity in bacteria than in the fungi associated with the rhizosphere. Currently, we are working on determining the effect of the plant genotype on the establishment of beneficial microbial communities to promote better management of biological processes such as plant nutrition, pest control, and biotic interactions.

P87: Transcription factors and regulation of aquaporin expression in maize stomatal guard cells and subsidiary cells

Biochemical and Molecular Genetics Sanskriti Vats (Postdoc)

Vats, Sanskriti1
Ding, Lei2
Laurent, Maxime1
Chaumont, François1

1Louvain Institute of Biomolecular Science and Technology, UCLouvain, 1348 Louvain-la-Neuve, Belgium
2Earth and life institute, UCLouvain, 1348 Louvain-la-Neuve, Belgium

Grasses display rapid and efficient stomatal movements, contributing to superior water-use efficiency and photosynthetic capacity. This performance arises from the unique organization of grass stomata, where dumbbell-shaped guard cells (GCs) interact with flanking subsidiary cells (SCs) to coordinate solute and water fluxes. Although aquaporins (AQPs) are critical for facilitating water and small solute transport across membranes, their specific and potentially divergent roles in GCs and SCs remain largely unexplored. Recent cell-specific transcriptomics analyses revealed distinct GC- and SC-specific expression patterns of several AQP isoforms in maize. Interestingly, GC-specific CRISPR/Cas9 knock out of the plasma membrane AQP genes belonging to the PIP1subfamily enhanced stomatal opening and slowed stomatal responses to light and CO₂, compared to wild-type plants. In parallel, a genome-wide association study for the expression of AQP genes in leaves of 254 maize lines, eQTL mapping and regulatory network analyses identified transcription factors (TFs) associated with AQP expression variations, several expressed in GCs and/or SCs. We are currently dissecting how these TF–AQP modules regulate maize stomatal behavior and contribute to grass-specific stomatalkinetic efficiency. We will validate the ability of candidate TFs to modulate AQP expression using transactivation assays, EMSA-based promoter binding analyses, and engineered maize suspension cell systems. To assess their in vivo function, we are using GC- and SC-specific CRISPR/Cas9-mediated knockouts and targeted overexpression of both AQPs and validated TFs. The resulting lines will be examined at both cellular and whole-plant levels for stomatal movements. Integrating molecular, cellular, and physiological approaches will uncover how cell-specific AQP regulation shapes stomatal performance in grasses.

P88: Understanding expression dynamics of flowering time genes from a gene network perspective

Biochemical and Molecular Genetics Joseph DeTemple (Graduate Student)

DeTemple, Joseph M1
Hinrichsen, Jacob T1
Li, Dongdong1
Schoenbaum, Gregory R1
O'Rourke, Jamie A1 2
Graham, Michelle A1 2
Li, Xianran3
Vollbrecht, Erik4
Muszynski, Michael5
Yu, Jianming1

1Department of Agronomy, Iowa State University, Ames, IA, US
2USDA-ARS Corn Insects and Crop Genetics Research Unit, Ames, IA, US
3USDA-ARS, Wheat Health, Genetics, and Quality Research, Pullman, WA, US
4Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA, US
5Department of Tropical Plant and Soil Sciences, University of Hawai’i at Mānoa, Honolulu, HI, US

Flowering time is an important adaptive trait for maize plants to respond to environmental signals like photoperiod. This trait is controlled by a network of genes that capture, process, and interpret these signals from leaves to the apical meristem. In this study, we set out to answer three questions about the genes within this network: 1) which genes are most important for explaining flowering time differences between genotypes, 2) which developmental stages are most critical for each gene, and 3) which timepoints are most important for capturing expression differences for each gene. To answer these questions, we sampled leaf tissue from four temperate (B73, Mo17, Oh43, and TX303) and four tropical (CML277, CML52, Ki3, and Tzi8) maize inbreds under long- and short-day conditions in growth chambers. To maximize our sampling space, we ran over 12,000 RT-qPCR reactions to determine expression of eight genes spread across the circadian clock and photoperiod pathways (cca1, cct1, col3, elf3.1, gi1, pebp8, prrtf1, and toc1) across three timepoints (morning, midday, and night) and across five v-stages (V2-V10). Data was modeled using a linear mixed model approach with a random effect for growth chamber and fixed effects for gene, genotype, photoperiod, timepoint, and v-stage. We find that the genes cct1 and cca1 have the highest effect when modeling expression, reinforcing their role as major flowering time regulators. Interestingly, we also found that the most predictive timepoints for each gene were generally not the timepoints with the highest expression. These findings provide new insights into the dynamics of the flowering time gene network and the connections between genetic variation, gene expression, and flowering time. Interpreting these patterns in the context of the overall flowering time gene network is critical for understanding how both newly discovered and classical genes interact in the genetic regulation of flowering time.

P89: Xylem sap proteomics reveals defense mechanisms underlying pierce’s disease tolerance in grapevines

Biochemical and Molecular Genetics Ramesh Katam (Principal Investigator)

Katam, Ramesh1
Cardillo, Madison1 2
Weaver-Johnson, Weaver-Johnson1

1Florida A&M University, Tallahassee FL USA 32307
2Florida State University, Tallahassee FL USA 32306

Pierce’s disease (PD), caused by the bacterium Xylella fastidiosa, severely impacts grapevine cultivation worldwide. The disease is transmitted by sharpshooters that feed on xylem sap, leading to vessel blockage and plant wilting. While Vitis vinifera is highly susceptible to PD, muscadine grapes (Vitis rotundifolia) and Florida hybrid bunch cultivars exhibit significant tolerance. This study explores the proteomes of xylem tissue and xylem sap from PD-susceptible Vitis vinifera, PD-tolerant muscadine, and Florida hybrid bunch cultivars to better understand the molecular mechanisms underlying PD tolerance. Using comprehensive LC-MS/MS analysis, we identified around 2000 proteins in xylem tissue and 400 proteins in xylem sap. Key defense-related proteins, including beta-1,3-glucanase, peroxidase, WD repeat-containing proteins, and receptor-like protein kinases, were abundantly expressed in the xylem tissue and sap of muscadine and Florida hybrid cultivars, but were found in lower levels in Vitis vinifera. These proteins, which play roles in pathogen defense and oxidative stress response, are potentially involved in the disease tolerance mechanisms of muscadine and Florida hybrid cultivars. Our findings suggest that the enhanced expression of defense proteins in xylem sap may contribute to the resistance of these cultivars to Xylella fastidiosa, providing valuable targets for future breeding strategies aimed at improving PD tolerance in grapevines.

P90: ZmWAKL-ZmCPK39 synergistic immunity drives super-additive resistance to gray leaf spot in maize

Biochemical and Molecular Genetics Mingliang Xu (Principal Investigator)

Guo, Chenyu1
Zhu, Mang1
Zhong, Tao1
Li, Qian1
Xu, Mingliang1

1State Key Laboratory of Maize Bio-breeding/College of Agronomy and Biotechnology/National Maize Improvement Center, China Agricultural University, Beijing 100193, P. R. China

Gray leaf spot (GLS), caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina, is a major foliar disease of maize (Zea mays L.) worldwide. We previously identified two immune pathways conferring GLS resistance: a ZmWAKL-ZmWIK-ZmBLK1-ZmRBOH4 phosphorelay that drives reactive oxygen species (ROS) bursts, and a ZmCPK39-ZmDi19-ZmPR10 module that regulates production of the pathogenesis-related protein PR10. Here, we demonstrate that combining resistant alleles of ZmWAKL and ZmCPK39 generates super-additive immunity, with disease reduction exceeding combinatorial predictions. Mechanistically, ZmCPK39 dampens ROS via two routes: (1) phosphorylating ZmBLK1 at Ser89 to block ZmRBOH4 activation, and (2) directly targeting ZmRBOH4 for degradation. In parallel, ZmWIK facilitates ZmCPK39-dependent degradation of ZmDi19, thereby reducing PR10 accumulation. Pathogen-induced activation of ZmWAKL triggers ROS bursts that in turn induce ZmCPK39, establishing a negative feedback loop that restrains ROS production. Importantly, the resistant ZmCPK39 allele, characterized by lower inducible expression, enhances immunity by amplifying ROS bursts while alleviating PR10 suppression. This dynamic crosstalk between ROS and PR10 pathways maximizes immune output and provides a genetic framework for engineering durable GLS resistance.

Cell and Developmental Biology

P91: Discovering the roles of feronia-like receptor kinase genes in maize

Cell and Developmental Biology Lander Geadelmann (Graduate Student)

Geadelmann, Lander1 2
Unger-Wallace, Erica1
Malik, Shikha3
Wang, Ping1
Choi, Hyunho1 2
Yin, Yanhai1
Walley, Justin3
Guo, Hongqing1 2
Vollbrecht, Erik1 2

1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011
2Interdepartmental Plant Biology Program, Iowa State University, Ames, IA 50011
3Department of Plant Pathology, Entomology and Microbiology, Iowa State University, Ames IA 50011

Understanding at the molecular level how plants balance growth under favorable and diverse stress conditions can help develop strategies for engineering resilient crops. The FERONIA receptor kinase in Arabidopsis has been demonstrated to regulate many downstream signaling components to modulate plant growth and development, reproduction and stress responses. Despite involvement in these critical processes, characterization of the growth and development of feronia-like receptor (flr) kinase mutants remains largely unexplored in maize. Using CRISPR-cas9 technology, we generated mutants in three maize flr genes. While single-gene flr mutants are largely unaffected, higher-order mutants show slower growth, cell wall defects, and defective kernel phenotypes, but unaltered gametophyte transmission efficiencies, compared to their wildtype siblings. We produced a multi-omics dataset with a goal of identifying direct substrates of maize FLR kinases and to uncover growth and environmental stress response pathways they modulate. Putative substrates will be validated using an in vitro maize FLR kinase assay; mutants of these candidates will be crossed with flr alleles to investigate potential genetic and phenotypic interactions.

P92: Maize transcription factors alter meristem size and shoot architecture

Cell and Developmental Biology Lander Geadelmann (Graduate Student)

Geadelmann, Lander1
Unger-Wallace, Erica1
Strable, Josh1
Vollbrecht, Erik1

1Iowa State University; Genetics, Development, and Cellular Biology Department; Ames, IA, United States

The Shoot Apical Meristem (SAM) serves as a pool of indeterminate cells responsible for initiating leaves and the stem. Considerable genotype-dependent variation in SAM size and shape has been described in the literature, yet all maize ultimately follows the same shoot architectural pattern: leaves spaced approximately evenly up the plant, positioned in an alternating phyllotactic pattern. Thus, the genetic programming to produce this pattern operates robustly across a range of native SAM sizes. Mutants that perturb SAM size can disrupt architectural patterning to varying degrees. When the maize transcription factor EREB130 and related genes are mutated, the meristem enlarges, phyllotactic patterning can be disrupted, and the stem becomes kinked and compressed. Mutants of FASCIATED EAR4 (fea4), a bZIP transcription factor, also show an enlarged SAM, but in B73 its effects on shoot architecture are subtle (Pautler 2015). Both mutants’ phenotypes are enhanced in the ereb130;fea4 double mutant combination. Similarly, FASCIATED EAR3 (fea3) encodes a transmembrane receptor expressed in the SAM whose mutant also enlarges the SAM (Je 2016) without causing a discernible shoot architecture phenotype; again, phenotypes are enhanced in the ereb130;fea3 double mutant. Finally, we also examined double mutants of ereb130 and knotted1 (kn1), a homeobox transcription factor that reduces SAM size (Vollbrecht 2000). We found that kn1’s reduction in SAM size is epistatic to ereb130’s enlargement, yet kn1’s normal shoot architecture is not fully restored. FEA4 and FEA3 are known to act in separate genetic pathways, and KN1 in a different but overlapping one, all affecting SAM size. Our results indicate that while SAM size alone does not explain changes in shoot architecture, spatiotemporal interactions between gene expression and SAM size can. To further explore these dynamics, higher-order mutants are being made between ereb130 and mutants of both SAM-size genes from the literature and candidate genes from experiments.

P93: A LITTLE ZIPPER–ROLLED LEAF1 regulatory module controls polarity in maize inflorescences

Cell and Developmental Biology Zongliang Chen (Postdoc)

Chen, Zongliang1
Galli, Mary1
Strable, Josh3
Bang, Sohyun4
Schmitz, Robert4
Gallavotti, Andrea1 2

1Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ, USA
2Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA
3Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA USA
4Department of Genetics, University of Georgia, Athens, GA, USA

Apical–basal polarity in meristems is well established and maintained throughout plant development, in part through the HAM–WUS–CLV pathway. Within this module, WUS activates CLV3 expression in the apical domain, while the HAM–WUS suppresses CLV3 in the basal domain, restricting CLV3 to the apical region of the meristems. However, the mechanisms governing basal domain positioning remain poorly understood. We have identified a novel regulatory module composed of LITTLE ZIPPER (ZPR) and ROLLED LEAF1/REVOLUTA (RLD1/REV) that directs basal domain positioning beneath the apical stem cell domain. Previously, we demonstrated that the maize dominant mutant Barren inflorescence3 (Bif3) exhibits ZmWUS1overexpression in an unusual ring-shaped pattern within ear primordia, representing a striking shift from apical–basal polarity to radial symmetry. Here, we show that in Bif3, ZmWUS1 modulates the expression of basal-domain genes ZmZPR3, ZmZPR4a and ZmZPR4b in a corresponding ring-shaped pattern, and triple knockouts of ZmZPR3;4a;4b partially restore Bif3 inflorescence phenotypes. Single-cell ATAC-seq analysis revealed that ZmZPR4b possesses a more accessible proximal promoter region in ZmWUS1-expressing cells of Bif3 ear primordia compared to wild-type ears, suggesting that ZmZPR3/4a/4b ectopic expression contributes to the inflorescence meristem rearrangement observed in Bif3. Yeast two-hybrid assays, AlphaFold structural predictions, and DAP-seq experiments indicate that ZmZPR3/4a/4b physically interact with RLD1 via their ZIP domains, preventing RLD1 homodimerization and thereby inhibiting its DNA-binding activity. CRISPR-Cas9-mediated triple knockout of RLD1 and its closest paralogs RLD2 and RLD3 produced Bif3-like ear defects, including shortened ears with primordia lacking a discernible inflorescence meristem. Together, with their overlapping expression patterns, these findings support a model in which ZmWUS1 misexpression in Bif3ears expands the expression domains of inhibitory ZPRs within the apical domain and restrain RLD function in the stem cell niche. This reveals a previously unrecognized ZPR–RLD1 regulatory module governing stem cell organization in maize inflorescences.

P94: A Tri-peptide signaling mechanisms regulates pollen tube growth and guidance during double fertilization in maize

Cell and Developmental Biology Xia Chen (Postdoc)

Chen, Xia1
Zhou, Liangzi1
Uebler, Susanne1
Dresselhaus, Thomas1

1Plant Cell Biology, Biochemistry, and Biotechnology; University of Regensburg; Regensburg, Germany, 93053

Pollen tube growth and guidance are essential steps for successful double fertilization in flowering plants. In monocot grasses such as maize, these processes occur within specialized reproductive structures and rely on precise cell-cell communication between the male gametophyte (pollen/pollen tube) and the female tissues such as papillar hair cells, transmitting tract, and egg apparatus. In maize, pollen grains hydrate and germinate rapidly on the papilla hair cells of silk and elongated pollen tubes grow through the transmitting tract toward the ovule. Upon reaching the micropylar region, pollen tubes must respond to local attractant signals from the female gametophyte to penetrate the nucellus and enter the embryo sac. A small protein or peptide specifically expressed in the egg apparatus named Zea mays EGG APPARATUS1 (ZmEA1) has been reported as a pollen tube attractant acting in a species-specific manner. How ZmEA1 mediates this guidance process has remained unclear. Using affinity purification combined with mass spectrometry and phosphoproteomic analyses, we identified the pollen expressed small protein ZmWSL, and FERONIA-like receptor kinases (ZmFERLs) as direct interaction partners of ZmEA1. Our results suggest that these components form a functional signaling complex that mediates pollen tube perception and directional growth toward the embryo sac. We further show that the small cysteine-rich peptides ZmRALF2/3 regulate pollen tube growth dynamics and cell wall properties through the conserved RALF-LLG-CrRLK1L/LRX pathway. Our ongoing work focuses on integrating ZmRALF-, ZmWSL-, and ZmEA- mediated signaling to construct a unified molecular framework for pollen tube growth and guidance in maize. Together, these findings reveal a peptide-receptor interaction network that coordinates pollen tube navigation and fertilization in monocot crops.

P95: A nuclear role of RAMOSA3 in inflorescence branching independent of its enzymatic function

Cell and Developmental Biology Xiaosa Xu (Principal Investigator)

Xu, Xiaosa1 2
Wang, Zhen1
Tran, Thu2
Claeys, Hannes2 4
Lang Vi, Son2 5
Zhang, Yasmine1
Chuck, George3
Jackson, David2

1Department of Plant Biology, University of California, Davis, CA 95616, USA
2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
3Plant Gene Expression Center, UC Berkeley, Albany, CA, 94710, USA
4Current address: Inari, Industriepark 7A, 9052 Zwijnaarde, Belgium
5Current address: Agricultural Genetics Institute, Hanoi, Vietnam

Plant development emerges from stem cell populations called meristems, which control organ initiation and branching. RAMOSA3 (RA3), a classical maize developmental gene, controls inflorescence branching, and encodes a trehalose phosphate phosphatase enzyme. Our recent genetic and cell biology studies found that RA3 has a potentially non-enzymatic moonlighting function, since its phenotype can be uncoupled from catalytic activity. Furthermore, RA3 protein forms nuclear speckles, suggesting that it is associated with transcriptional regulatory machinery in the nucleus. To tackle the mystery of the nuclear moonlighting function of RA3, we performed ethyl methyl sulfonate (EMS) mutagenesis and screening of ra3 mutants, and identified an enhancer, indeterminate spikelet1 (ids1). IDS1 is an AP2 type transcription factor that controls spikelet and floret development. By carefully examining the early developmental stages of ra3;ids1 double mutants, we found that floral meristems were transformed into branches, rather than forming florets. We confirmed these findings by crossing ra3 with additional ids1 alleles and conducting allelism tests. Using in-situ hybridization, we found RA3 and IDS1 were co-expressed in the boundary regions between floral meristems, which was also supported by our single-cell transcriptomic profiling data. To further examine if IDS1 might be involved in the hypothetical transcriptional regulatory function of RA3, we checked for physical interactions and colocalization between RA3 and IDS1. Indeed, we found that RA3 and IDS1 proteins interact in the nucleus and form speckles in planta. In vivo co-immunolocalization using native RA3 and IDS1 antibodies and super-resolution microscopy confirmed their co-localization in nuclear speckles at the boundary regions between maize floral meristems. Through RNA-seq analysis of ra3 and ids1 single and double mutants, we identified downstream candidate genes, including a serine protease–encoding gene, that are co-regulated by the putative RA3–IDS1 complex. Together, our data suggests that RA3 had a nuclear regulatory role in controlling inflorescence branching by interacting with the transcription factor, IDS1.

P96: A receptor like kinase-heterotrimeric G protein module regulates immune responses in maize

Cell and Developmental Biology Shujun Meng (Postdoc)

Meng, Shujun1 2
Wu, Qingyu1 2

1Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China, 100081
2National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, China, 572024

Abstract: During crop growth and development, plants must continually defend against pathogen infections; however, there is a tradeoff between disease resistance and development, as excessive immune activation often suppresses growth and leads to yield reduction. Thus, unraveling the growth-defense balance network is crucial for breeding high-yield, disease-resistant cultivars. The heterotrimeric G proteins in maize serve as a critical hub regulating inflorescence development and disease resistance immunity. However, the molecular regulatory networks through which they mediate development and immunity remain unclear, limiting the rational manipulation of G-protein signaling to achieve a development–immunity balance. Here, we show that homozygous Zmgb1 loss-of-function mutants exhibit early seedling lethality caused by severe autoimmunity. Through an EMS suppressor screen, we identified a mutant that partially rescues the Zmgb1 lethal phenotype. Map-based cloning revealed that candidate gene encodes a leucine-rich repeats receptor-like kinase (LRR-RLK), thus named it as ZmRLKX1. Structural modeling indicated that the mutation induced a significant conformational change in the ATP-binding pocket of the kinase domain. Consistently, in vitro kinase assays demonstrated that the wild-type ZmRLKX1 possesses autophosphorylation activity, whereas the kinase-dead mutant completely lacks kinase activity. This study reveals that ZmRLKX1 regulates the ZmGB1-mediated development-immunity balance in a kinase activity‑dependent manner and provides insights into developmental and immune signaling pathways are coordinated in maize. These results offer new strategies and valuable germplasm resources for simultaneously improving both disease resistance and crop yield. Keywords: immune response, growth-defense tradeoff, LRR-RLK, heterotrimeric G protein

P97: A strategy for synthetic biology of large genome grasses

Cell and Developmental Biology Lukas Evans (Lab Manager)

Evans, Lukas J.1
Wu, Hao1 2
Hogue, Hayden1
Char, Si Nian3
Yang, Bing3
Scanlon, Michael J.1

1School of Integrative Plant Science, Cornell University, Ithaca, NY, USA 14853
2College of Agriculture, Nanjing Agricultural University, Jiangsu, Nanjing, CN 210095
3Division of Plant Sciences, University of Missouri, Columbia, MO, USA 65211

Grass leaves comprise a distal, photosynthetic blade and a proximal, wrapping sheath base. Ligule, collar, and auricle tissues develop at the blade-sheath boundary. The ligule is a fringe structure that adheres to the next inner leaf to block debris from entering the whorl. The hinge-like collar allows the leaf blade to bend away from the stem. Outgrowths from the base of the collar are called auricles, which clasp the leaf-base toward the stem. Ligules may be epidermally-derived in maize, vascularized L1-L2-derived in rice (Oryza sativa), rows of simple hair-like projections in Setaria viridis, or absent as in Echinochloa crus-galli. Maize lacks auricles, while they are prominent in many other cereals. Despite this morphological diversity, grasses exhibit extensive synteny and conservation of gene function, regardless of genome size or phylogenetic distance. For example, orthologous mutations in LIGULELESS1 (LG1), a gene responsible for blade-sheath boundary development, present nearly identical phenotypes in all grasses yet examined. Cis-regulatory element variation between orthologs may explain how similar genes produce different grass phenotypes. Our goal is to use grasses as a single genome to confer new ligule/auricle morphologies and explore the evolution of novel traits. Spatial transcriptomics will identify candidate genes controlling ligule and/or auricle variation between grasses. Promoters from small genome (1 Gbp) grasses. Conserved non-coding sequences are identified in BdWOX3 in Brachypodium distachyon and ZmNARROWSHEATH1 (ZmNS1) in maize. A BdWOX3 homologous promotor was engineered to drive ZmNS1 fused to YFP to observe protein localization. BdWOX3p:NS1-YFP protein accumulation corresponds to mRNA in situ hybridizations of ZmNS1 transcripts. Complementation tests are ongoing. These experiments provide a strategy for synthetic biology approaches toward crop improvement in large genome grasses.

P98: A trypsin-like serine protease ZmNAL1a fine-tunes maize floral transition and flowering time

Cell and Developmental Biology Lei Liu (Principal Investigator)

Li, Nan1
Ning, Qiang2
Li, Zhen（真）1 3
Zhang, Qian1
Zhu, Peilu4
Zhan, Jimin1
Li, Yunfu1
Li, Zhen（震）1
Dong, Liang1
Xiong, Qing1
Liao, Jiahao4
Liu, Jie3
Jackson, David5
Kitagawa, Munenori4
Zhang, Zuxin1 3
Liu, Lei1

1National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, China
2Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
3Yazhouwan National Laboratory, Sanya 572024, China
4National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China
5Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

The floral transition is a crucial phase in flowering plants that initiates reproductive development. Florigen, a key regulator of this transition, is expressed in the leaves and transmits environmental signals by trafficking to the vegetative shoot apical meristem, thereby promoting the floral transition. However, whether additional signals, expressed outside the meristem control the flowering transition remains to be explored. This study identified another floral transition signal, ZmNAL1a, which encodes a trypsin-like serine protease and can move from the leaf to the shoot apical meristem via plasmodesmata to regulate floral transition in maize. Mutation of ZmNAL1a suppresses the expression of key flowering genes in the shoot apical meristem, resulting in a delayed floral transition and flowering. ZmNAL1a interacts with and degrades RAMOSA1 ENHANCER LOCUS2 (REL2), a TOPLESS-like corepressor, which can regulate the expression of flowering genes by affecting histone acetylation and transcriptional regulation alongside ZmEREBP147, an AP2/EREBP transcription factor. These findings suggest that ZmNAL1a is a diffusible signal that regulates the floral transition and flowering via a conserved NAL1-TOPLESS epigenetic regulation module and through transcriptional regulation. This discovery broadens the understanding of flowering control, offering potential targets for improving adaptation and crop yield through precise manipulation of flowering time.

P99: ARFTF17 regulates dent and flint kernel architecture in Maize

Cell and Developmental Biology Haihai Wang (Research Scientist)

Wang, Haihai1 8
Huang, Yongcai1 7 8
Li, Yujie1 2 8
Cui, Yahui1
Xiang, Xiaoli1
Zhu, Yidong1 2
Wang, Qiong1
Wang, Xiaoqing4
Ma, Guangjin1 2
Xiao, Qiao1 2
Huang, Xing1 2
Gao, Xiaoyan1
Wang, Jiechen1
Lu, Xiaoduo5
Larkins, Brian A.6
Wang, Wenqin3 9
Wu, Yongrui1 9

1State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology &Ecology, Chinese Academy of Sciences, Shanghai 200032, China
2University of the Chinese Academy of Sciences, Beijing 100049, China
3Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
4Forestry and Pomology Research Institute, Shanghai Academy of Agriculture Sciences, Shanghai 201403, China
5Institute of Molecular Breeding for Maize, Qilu Normal University, Jinan 250200, China
6School of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA
7State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, China
8These authors contributed equally
9Correspondence should be addressed to Y.W. (yrwu@cemps.ac.cn) and W.W. (wang2021@shnu.edu.cn)

Dent and flint kernel architectures are important characteristics that affect the physical properties of maize kernels and their grain end uses. The genes controlling these traits are unknown, so it is difficult to combine the advantageous kernel traits of both. We found mutation of ARFTF17 in a dent genetic background reduces IAA content in the seed pericarp, creating a flint-like kernel phenotype. ARFTF17 is highly expressed in the pericarp and encodes a protein that interacts with and inhibits MYB40, a transcription factor with the dual functions of repressing PIN1 expression and transactivating genes for flavonoid biosynthesis. Enhanced flavonoid biosynthesis could reduce the metabolic flux responsible for auxin biosynthesis. The decreased IAA content of the dent pericarp appears to reduce cell division and expansion, creating a shorter, denser kernel. Introgression of the ARFTF17 mutation into dent inbreds and hybrids improved their kernel texture, integrity, and desiccation, without affecting yield.

P100: ARFTF17 regulates dent and flint kernel architecture in Maize

Cell and Developmental Biology Yidong Zhu (Graduate Student)

Wang, Haihai1 8
Huang, Yongcai1 7 8
Li, Yujie1 2 8
Cui, Yahui1
Xiang, Xiaoli1
Zhu, Yidong1 2
Wang, Qiong1
Wang, Xiaoqing4
Ma, Guangjin1 2
Xiao, Qiao1 2
Huang, Xing1 2
Gao, Xiaoyan1
Wang, Jiechen1
Lu, Xiaoduo5
Brian A, Larkins6
Wang, Wengin3 9
Wu, Yongrui1 9

1State Key Laboratony of Plant Trat Design, CAs Center for Excelencein Molecuar Plant sciences,shanghai insiute o Plant Physiology &cology, chinese Academy of sciencesShanghai 200032, China
2University of the Chinese Academy of Sciences, Beijing 100049, China
3Shanghai Key Laboratory of Plant Molecular Sciences, college of Life Sciences, Shanghai Normal University, Shanghai 200234, China
4Forestry and Pomology Research Institute, Shanghai Academy of Agriculture $ciences, Shanghai 201403, China
5institute of Molecular Breeding for Maize, Qilu Nommal University, Jinan 250200, China
6School of Plant Sciences, University ofArizona, Tucson, Arizona 85721, USA
7State Key Laboratory of Crop Gene Exploraion and Utlization in Southwest China, Sichuan Agricutural Universily, Chengdu 61130, China
8These authors contributed equally
9corespondence should beaddressed to yW (yrwu@cemos ac cn) and Ww (wan02021@shnu edu.cn)

Dent and flint kernel architectures are important characteristics that affect the physical properties of maize kernels and their grain end uses. The genes controlling these traits are unknown, so it is difficult to combine the advantageous kernel traits of both. We found mutation of ARFTF17 in a dent genetic background reduces IAA content in the seed pericarp, creating a flint-like kernel phenotype. ARFTF17 is highly expressed in the pericarp and encodes a protein that interacts with and inhibits MYB40, a transcription factor with the dual functions of repressing PIN1 expression and transactivating genes for flavonoid biosynthesis. Enhanced flavonoid biosynthesis could reduce the metabolic flux responsible for auxin biosynthesis. The decreased IAA content of the dent pericarp appears to reduce cell division and expansion, creating a shorter, denser kernel. Introgression of the ARFTF17 mutation into dent inbreds and hybrids improved their kernel texture, integrity, and desiccation, without affecting yield.

P101: Advances at the Wisconsin Crop Innovation Center to enable maize research

Cell and Developmental Biology Dayane Cristina Lima (Research Scientist)

Lima, Dayane Cristina1
Lor, Vai1
Walter, Nathalie1
Collier, Ray1
Mahoy, Jill1
Hahn, Emily1
McGinty, Alyson1
Kaeppler, Shawn1 2

1Wisconsin Crop Innovation Center, University of Wisconsin-Madison, 8520 University Green, Middleton, WI 53562
2Department of Plant and Agroecosystem Sciences, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706

The Wisconsin Crop Innovation Center (WCIC - cropinnovation.cals.wisc.edu) at the University of Wisconsin–Madison is a public, fee-for-service plant transformation center, providing advanced genetic transformation and gene editing services to academic and private-sector clients. WCIC maintains high stewardship and quality management standards with Excellence Through Stewardship (ETS) and the Plant Breeding Innovation Management Program (PBIMP) certifications. The center supports a broad range of crops, including maize and soybean, and delivers end-to-end solutions that bridge molecular innovation and field-ready applications.The center’s activities are divided between fee-for-service and Research and Discovery (R&D) and have supported clients worldwide in achieving their research objectives. In maize, diverse projects involves trait introgression and transformation and deliver research-ready T₂ seed for testing across environments. WCIC carries out Agrobacterium-mediated transformations across a diverse set of genetic backgrounds, including elite temperate lines such as B73, LH244, LH287, PH1CA, PH2MW, PH09B, as well as sweet corn and tropical germplasm. In one recent project, WCIC designed more than 100 transformation vectors targeting classical maize genes. The targeted genes influence key aspects of plant architecture, environmental responsiveness, and traits that directly or indirectly affect yield (grain and biomass). Information on the outcomes and workflow will be presented.Projects supported by WCIC include the Circular Economy for Reimagining Corn Agriculture (CERCA). CERCA focuses on redesigning corn production systems to improve nitrogen utilization and reduce fertilizer inputs while maximizing starch yield. WCIC supports CERCA by delivering gene-edited maize seed ready for trialing, targeting, for example, genes involved in cold resilience and regulation of the photosynthetic machinery. Efficient processes advanced through this multi-investigator collaboration will be discussed as an example of moving from hypothesis to research-ready materials.

P102: An α-L-fucosidase WILTING LEAF 1 maintains maize abiotic stress resistance by regulating vascular development

Cell and Developmental Biology Zichao Li (Graduate Student)

Zichao, Li1
Changxiong, Ke1
Lu, Kang1
Liang, Dong1
Lei, Liu1
Fang, Yang1 2
Wanshun, Zhong1 3

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China, 430070
2School of Agriculture and Biotechnology, Sun Yat-Sen University, Shenzhen, China, 518107
3Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China, 518120

Maize (Zea mays) yield is often limited by diverse abiotic stresses, such as heat and drought. In this study, we identified and characterized a novel maize mutant, wilting leaf1 (wl1-ref), which exhibits leaf wilting, dwarfism, and impaired production. The wilting phenotype was aggravated under multiple abiotic stresses, including high temperature, drought, and salt conditions. wl1-ref showed severe defects in vascular bundle development with abnormal or absent protoxylem vessel, leading to obvious compromise water transport. WL1 encodes an α-L-fucosidase from the GDSL family, localized to the endoplasmic reticulum, and was highly expressed in vascular-rich tissues including roots, stems, and leaf sheaths. Transcriptome profiling revealed substantial changes in the expression of cell wall biosynthesis-related genes, along with reduced lignin content and disrupted cell wall architecture. Taken together, our results demonstrate that WL1 modulates water transport and abiotic stress tolerance in maize by affecting cell wall integrity and vascular development, highlighting its potential as a target for breeding stress-resilient maize varieties.

P103: Antagonistic transcription factors regulate antisymmetric wrapping in grass leaves

Cell and Developmental Biology Mike Scanlon (Principal Investigator)

Ragas, Richie Eve G.1
Cheng, Jie2
Evans, Lukas J1
Palos, Kyle3
Nelson, Andrew D. L.3
Agha, Kayden1
Specht, Chelsea D.1
Smith, Richard S.2
Coen, Enrico4
Scanlon, Michael J1

1School of Integrative Plant Science, Cornell University, Ithaca, NY, USA 14853
2Department of Computational and Systems Biology, John Innes Centre, Norwich, UK NR4 7UH
3Boyce Thompson Institute, Cornell University, Ithaca, NY, USA 14853
4Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK NR4 7UH

Antisymmetry describes a type of bilateral asymmetry where left- or right-handedness occurs equally within populations. The overlapping leaf bases of grasses present an asymmetric left-right pattern where the outer (wrapper) margin surrounds the inner (tucker) margin. Left-right wrapper-tucker orientation is perpetuated non-randomly throughout all leaves in the plant, yet the handedness of this pattern, wrapper on the left or on the right, is equally-distributed among individuals. To better understand the mechanisms of antisymmetric leaf patterning, we used higher-order genetics, multiplexed transcriptomics, and computational simulations to inform a model where antagonistic interactions of plant transcription factors regulate non-random, non-equivalent growth and boundary formation in the developing wrapper-tucker margins of grass leaves. WUSHEL-like HOMEOBOX3a (WOX3a), a transcription factor implicated in marginal growth, accumulates asymmetrically in the emerging wrapper whereas the boundary gene CUP-SHAPED COTYLEDON2 (CUC2) is enriched in the tucker and promotes margin separation. Genetic analyses indicate that LIGULESS1 (LG1) represses WOX3a expression in the tucker; DNA Affinity Purification sequencing data suggest that CUC2 and WOX3a are mutually repressive. Higher-order wox3 mutants exhibit aberrant, random wrapper -tucker patterning. The data suggest that in both plants and animals, morphological asymmetry ensues via the unequal, left-right accumulation of antagonistic factors. @font-face {font-family:“Cambria Math”; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:““; margin:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:”Arial”,sans-serif; mso-fareast-font-family:Arial; mso-ansi-language:EN;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:11.0pt; mso-ansi-font-size:11.0pt; mso-bidi-font-size:11.0pt; font-family:“Arial”,sans-serif; mso-ascii-font-family:Arial; mso-fareast-font-family:Arial; mso-hansi-font-family:Arial; mso-bidi-font-family:Arial; mso-font-kerning:0pt; mso-ligatures:none; mso-ansi-language:EN;}.MsoPapDefault {mso-style-type:export-only; line-height:115%;}div.WordSection1 {page:WordSection1;}

P104: Blurring the line: blade-sheath boundaries and the extended auricle1 mutant

Cell and Developmental Biology Heather Jones (Graduate Student)

Jones, Heather1
Ruggiero, Diana2
Evans, Lukas3
Scanlon, Michael J3
Leiboff, Samuel2
Richardson, Annis E1

1Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, EH9 3BF, UK
2Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA
3School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA

The maize leaf is neatly separated into distinct tissues along its proximodistal (base-to-tip, PD) axis: the proximal sheath wraps around the stem and provides structural support, and the distal blade bends away from the stem to intercept light. At the blade-sheath boundary, the specialised structures of the ligule and auricles arise, which regulate leaf angle. Leaf angle and shape are key agronomic traits, but how such a boundary is specified, and how these specialised structures form, is not well understood.The recessive extended auricle1 (eta1) mutant has a diffuse, shifted blade-sheath boundary and waves of excess auricle tissue along the blade margins. The eta1 phenotype suggests that boundary specification is disrupted, indicating that ETA1 may be a component of the gene regulatory network that defines the blade-sheath boundary. Here, I detail my work in characterising and mapping the mutation, as well as recent findings in novel patterning components and their implications for our understanding of maize architecture.

P105: Boundary issues: investigating the role of LIGULELESS 2 in maize leaf development

Cell and Developmental Biology Ed Bridge (Postdoc)

Bridge, Ed1
Sylvester, Anne2
Hake, Sarah3 4
Richardson, Annis1 4

1Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh
2University of Wyoming, Laramie, Wyoming, USA
3USDA PGEC, Albany, California, USA
4UC Berkeley, Berkeley, California, USA

Plant organ shape is intrinsically linked to its function. As these functions differ from organ-to-organ and species-to-species, there is a huge diversity of plant organ forms, but each deriving from amorphous groups of undifferentiated cells. To achieve these diverse organ shapes, developmental programs are directed by a series of patterning mechanisms and signals which influence tissue identity and growth. In grass species, like maize, proximal-distal (P-D) patterning in leaves gives rise to distinct sheath and blade regions, bounded by specialised auricle and ligule structures. These structures play a role in controlling the angle of the blade and act as a determinate of light interception, essential for photosynthetic sugar production and therefore, plant yield. Our research focuses on understanding the gene networks that control the blade-sheath boundary formation across grass species, with a focus on the BZIP transcription factor LIGULELESS 2 (LG2). In maize, mutants of LG2 have upright leaves due to a diffuse boundary region and defective auricle and ligule development. While LG2 is known to control the expression of the key ligule development gene, LIGULELESS 1 (LG1), less is known about how LG2 is itself regulated. Interestingly, despite the general expression of LG2 leaf tissues, LG2 protein localises specifically to the boundary region during early ligule development, which we hypothesise is driven by post-translational modification (PTM). To study the players involved in this potential PTM, we investigated the protein-protein interactions of LG2 in planta and identified candidate proteins including the E3 ligase, RING27, as well as the TASSELS ON UPPER EARS 1 (TRU1), a maize BOP homolog which is known to act as a sheath identity marker. These data could present a possible link between known P-D patterning factors in maize leaves and components of the ubiquitination pathway which could define LG2 protein localization.

P106: Characterization of a novel maize mutant with altered plant architecture

Cell and Developmental Biology Alberto Galeazzo Caffi (Graduate Student)

Caffi, Alberto Galeazzo1
Castorina, Giulia1 3
Pavan, Stefano2
Consonni, Gabriella1

1Department of Agricultural and Environmental Sciences (DiSAA), Università degli Studi di Milano, 20133 Milan, Italy
2Department of Soil, Plant and Food Sciences, Università degli Studi di Bari “Aldo Moro”, 70126 Bari, Italy
3Present address: CREA, Research Centre for Genomics and Bioinformatics, 29017 Fiorenzuola, Italy

The functional study of key regulatory factors facilitates a deeper comprehension of the molecular mechanisms and signaling pathways that determine the establishment of above-ground plant architecture. The dwarf-6 (d-6) mutant was identified in a W22 family following EMS treatment and subsequently propagated by backcrossing heterozygous plants to other inbred lines, over numerous generations. Whole genome re-sequencing of the mutant line identified a polymorphism in a locus encoding a DOF transcription factor, thus providing a promising entry point into the pathways controlling plant growth and leaf positioning. The mutant phenotype was investigated in segregating F2 populations. The presence of homozygous mutant siblings was identifiable at the coleoptile stage, as evidenced by a slight reduction in coleoptile elongation. Mutant defects were also evident at later stages, since mutants were characterized by reduced height, if compared to wild-type plants, and by the presence of a more upright leaf angle. Height diminution was attributable to a reduction in internode elongation, while the alteration in leaf angle was attributable to a defect in the development of the auricle, a region located at the boundary between the leaf blade and sheath.The genetic interaction between d-6 and mutants impaired in the synthesis of brassinosteroids (BRs) has also been explored. All BR loss-of-function mutants that have been characterized to date display a dwarf plant phenotype and an erect leaf phenotype, indicating a conserved role of BRs in modulating these traits. We will present the phenotypic evaluation of an F2 population segregating for d-6 and lilliputian1-1 (lil1-1), a recessive allele of the brassinosteroid-deficient dwarf1 (brd1) gene, encoding a brassinosteroid C-6 oxidase involved in the final steps of the pathway. The results obtained from this analysis indicate an additive effect of the two genes on the traits under study.

P107: Characterization of conserved growth promoting cytochrome P450 genes

Cell and Developmental Biology Büşra Çelik (Graduate Student)

Çelik, Büşra1 2
Villers, Timothy1 2
Beauchet, Arthur1 2
Nelissen, Hilde1 2

1Ghent University, Department of Plant Biotechnology and Bioinformatics, Technologiepark 927, 9052 Ghent, Belgium.
2VIB Center for Plant Systems Biology, Technologiepark 927, 9052 Ghent, Belgium.

The conserved growth promoting cytochrome P450 (CYP) 78A subfamily has a great potential for crop improvement, but its exact function is still unknown. In maize, a member of this family, called PLASTOCHRON1 (PLA1, ZmCYP78A1) is specifically expressed in the boundary of meristematic and differentiating cells in the maize shoot apical meristem, where cells divide more rapidly and enter the differentiation process. PLA1 stimulates organ growth, biomass and seed yield potential by extending the duration of cell division. This growth-promoting effect, along with the conserved expression profile, is maintained across diverse plant species. Therefore, we investigate CYP78As both in Arabidopsis (AtKLUH, CYP78A5) and maize (PLA1, ZmCYP78A1), taking advantage of the established research methodologies available for these species. We characterize CYP78As by determining their enzymatic activity, protein localization and protein interaction network, assessing their impact on hormonal responses and metabolic changes, and evaluating their possible contribution to short-range signaling. Our findings reveal novel insights into the role of CYP78As in fine-tuned regulation of organ growth in plants, opening new avenues for future research and applications.

P108: Characterization of roothairless 7 in maize

Cell and Developmental Biology Theo Nowak (Undergraduate Student)

Nowak, Theo1 2
Wichterich, Knut3
Hochholdinger, Frank1
Marcon, Caroline1 2

1INRES, Institute of Crop Science and Resource Conservation, Crop Functional Genomics, University of Bonn, 53117 Bonn, Germany
2INRES, Institute of Crop Science and Resource Conservation, BonnMu: Reverse Genetic Resources, University of Bonn, 53117 Bonn, Germany
3INRES, Institute of Crop Science and Resource Conservation, Horticultural Science, University of Bonn, 53121 Bonn, Germany

Root hairs are tubular extensions of epidermal cells. They are important for water and nutrient uptake from the soil by increasing the root surface. In maize (Zea mays L.), six roothairless mutants (rth1 – rth6) have been identified. Here, we report the characterization of the novel mutant rth7, which is affected in root hair elongation. We identified the rth7 mutant in a phenotypic screen of an F2 population derived from the Mutator-tagged BonnMu collection. Complementation crosses with rth1 – rth6 mutants are underway to determine allelism. Stereo microscopy demonstrated that rth7 mutants display significantly shorter root hairs than their wild-type siblings in all major root-types including primary-, seminal- and crown roots. Quantification of root hair length established that on average rth7 root hairs developed only 6 – 10% of the length of wild-type root hairs in primary-, seminal- and crown roots. Moreover, we analyzed root hair morphology of wild-type and rth7 at the seedling stage via ESEM (environmental scanning electron microscopy). High-resolution ESEM pictures revealed that rth7 forms morphologically normal but substantially shorter root hairs compared to the wild-type. The BonnMu F2 stock, segregating for the rth7 mutant, carries germinal Mutator insertions in 209 distinct genes, which makes it challenging to promptly identify the specific gene associated with the phenotype. We are currently prioritizing a candidate gene associated with the rth7 phenotype.

P109: Characterization of GRNs controlling SAM signaling centers

Cell and Developmental Biology Antje Feller (Postdoc)

Feller, Antje C1
Mondragon, Cecilia L1
Timmermans, Marja C1

1University of Tuebingen/ZMBP, Auf der Morgenstelle 32, Tuebingen, Germany

The Shoot Apical Meristem (SAM) is organized into functionally distinct domains defined by divergent molecular signatures (Knauer et al. 2019). Homeostasis in the SAM, which maintains the dynamic balance between stem cell self-renewal and the differentiation of daughter cells to form new organs, depends on signaling centers in the epidermis and the so-called organizing center. We are investigating the pathways controlling SAM homeostasis by characterizing Gene Regulatory Networks (GRNs) defining the signaling centers. We selected domain specific Transcription Factors (TFs) and performed RNA-seq analysis and Cleavage Under Targets and Tagmentation (CUT&tag) using a transient expression system in maize leaf protoplasts. This system delivers a reproducible pipeline for FACS, RNA-seq and CUT &Tag. Thus far we have analyzed x distinct TFs, the outcomes of which will be presented.

P110: Characterization of a novel embryo-defective mutant affecting embryo-endosperm development in maize

Cell and Developmental Biology Jiyun Go (Graduate Student)

Go, Jiyun1
Hong, Seongmin1
Park, Jinseong1
Lee, Hobin1
Seok, Myeong-geon1
Yi, Gibum1

1Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, Daejeon, Republic of Korea, 34134

Maize kernels primarily consist of the embryo and the endosperm, which interact bidirectionally during development. Genetic analyses of mutants, including emb (embryo-specific), dek (defective kernel), have demonstrated that coordinated embryo-endosperm development is essential for normal kernel formation. Endosperm-controlled nutrient allocation involving MEG1 and SWEET4c in the basal transfer layer (BETL), auxin-mediated embryo patterning, and embryo-derived signaling are all required for proper kernel growth and developmental progression. We identified a natural kernel mutant, emb-0404, from a Korean maize landrace, Sunchang Chal. The emb-0404 mutant exhibited abnormal embryo development accompanied by a shrunken endosperm phenotype. In the F2 population, the phenotype segregated in a 3:1 ratio, indicating control by a single recessive gene. To identify the causative gene, we separately pooled 30 wild-type (WT) and 30 mutant kernels and performed BSA-seq (bulk segregation sequencing). Allele frequency differences between the two bulks revealed a candidate region on chromosome 5L, and we aim to locate the causative locus by the map-based cloning approach. To date, no emb mutants have been reported within this region, suggesting that emb-0404 represents a novel mutant. Histological analyses were conducted to investigate embryo-endosperm interactions during kernel development. The emb-0404 mutant was confirmed to impair early stages of kernel development, with abnormal embryo development detectable as early as 12 days after pollination. Mutant embryos remained at a proembryo-like stage and were significantly smaller compared to WT embryos, which had differentiated shoot and root apical meristems. In addition, the mutant endosperm exhibited a floury and shrunken phenotype, supporting a functional interaction between embryo and endosperm development. Together, these results indicate that emb-0404 affects coordinated embryo-endosperm development, and identification of the underlying gene will provide insight into the genetic control of maize kernel development.

P111: Characterization of phenylboronic acid-responsive pathways in maize (Zea mays L.)

Cell and Developmental Biology Julia Brück (Undergraduate Student)

Brück, Julia1
Plotecki, Felix1
Chu, Liuyang1
Hochholdinger, Frank1
Matthes, Michaela S.1

1INRES - Crop Functional Genomics; Römerstraße 164; Bonn, Germany 53117

*Poster will be equally presented by Julia Brück and Felix PloteckiBoron is an essential micronutrient required for normal plant growth and development, taken up predominantly as boric acid. Boron deficiency causes defects in root development, including reduced primary root length and lateral root density. Boron primarily stabilizes the cell wall through cross-linking of the pectin sub-domain rhamnogalacturonan II (RG-II). Additional physiological roles of boron have been proposed but remain challenging to investigate. To handle this limitation, we utilize phenylboronic acid (PBA), a structural analogue of boric acid, which was hypothesized to chemically mimic boron deficiency by interference with RG-II. PBA treatment in maize (Zea mays L.) replicated and exceeded boron deficiency phenotypes. Strikingly, PBA did not interfere with RG-II dimerization, suggesting PBA-responsive pathways independent of RG-II. To uncover such pathways, we screened a population of 700 EMS-mutagenized maize lines (B73) and identified two lines appearing to produce an even more severe reduction of lateral root density and primary root length compared to B73 under PBA treatment. To characterize further developmental effects and potential lateral root initiation defects, these EMS mutant lines are phenotypically and histologically analyzed from seedling stage to maturity in greenhouse and field experiments. Moreover, both boron deficiency and PBA treatment decreased auxin and increased reactive oxygen species levels in primary roots. These findings indicate functional, potentially cell wall-independent crosstalk of boron and PBA with hormonal and redox pathways, which might mediate the observed phenotypic defects. Whereas exogenous application of H2O2 did not affect primary root growth, assays with the synthetic auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in lateral root density defects and reduced primary root growth. Moreover, the 2,4-D-induced primary root growth defects were partially restored when 2,4-D was applied together with PBA, emphasizing interactions of boron and PBA with auxin-mediated pathways. Our findings demonstrate that PBA can be a powerful tool to uncover boron-dependent processes in maize and suggest potential unidentified crosstalk between boron homeostasis and cell wall-independent pathways.

P112: Cold induced transcriptomic diversity in epidermis of maize different root types

Cell and Developmental Biology Fanghe Jin (Undergraduate Student)

Jin, Fanghe1
Zhou, Yaping1
Hochholdinger, Frank1

1INRES, Institute of Crop Science and Resource Conservation, Crop Functional Genomics, University of Bonn, 53117 Bonn, Germany

Cold stress adversely affects agricultural production worldwide in the context of global climate change. Due to its tropical origin, maize is particularly sensitive to low temperature stress. Root growth and functional responses to temperature determine the early establishment of plant vigour and subsequent growth. Epidermis exhibits a distinct transcriptomic cold response, with genes controlling root hair initiation and elongation dynamically regulated by temperature. Moreover, root-hair specific transcriptomic analyses further demonstrated a severity-dependent response: mild cold induces adaptive remodeling that maintains limited growth, whereas severe cold shifts transcriptional programs toward stress defense, resulting in pronounced morphological inhibition. The maize epidermis comprises tricoblasts and atricoblasts, of which only tricoblasts differentiate into root hairs. Along the longitudinal axis from the meristematic zone to the root hair zone, tricoblasts gradually acquire root hair identity, and their proportion relative to atricoblasts may vary across developmental zones. Yet the root type-specific and root zone-specific regulatory mechanisms of maize root in response to cold stress remain limited. Thus, we performed an epidermis-specific transcriptomic analysis to uncover the cold induced transcriptomic diversity in maize different root types. To this end, epidermal cells of three different root types from three developmental zones in maize under control and cold conditions were collected using laser capture microdissection. Subsequently, transcriptomic sequencing was performed followed by RNA isolated and quality control. We will perform differential gene expression analysis, functional enrichment analysis and co-expression network analysis to identify key regulators and pathways involved in the regulation of root hair plasticity under cold stress. Overall, our study will identify zone-specific regulatory mechanisms governing root hair developmental growth under cold stress and uncover candidate genes and pathways underlying root-type specific cold adaptation. Ultimately, the outcomes will provide new insights into the understanding of cold-induced regulation of root hair development in maize and may contribute to breeding strategies for improved cold resilience in cereals.

P113: Comprehensive phenotypic and transcriptomic analyses reveal multiple defects in the maize dwarf mutant dizzy1

Cell and Developmental Biology Xuelian Du (Graduate Student)

Du, Xuelian1 2
Klaus, Alina3
Lukas, Linnéa1 2
Alba, Magda Alejandra Guateque1 2
Stöcker, Tyll4
Carvajal, Jorge5
Zeisler-Diehl, Viktoria5
Schreiber, Lukas5
Schoof, Heiko4
Hochholdinger, Frank1
Marcon, Caroline1 2

1INRES, Institute of Crop Science and Resource Conservation, Crop Functional Genomics, University of Bonn, 53117 Bonn, Germany
2INRES, Institute of Crop Science and Resource Conservation, BonnMu: Reverse Genetic Resources, University of Bonn, 53117 Bonn, Germany
3AEI, Faculty of Agricultural, Nutritional and Engineering Science, Service Platform Plant Experiments, University of Bonn, 53115 Bonn, Germany
4INRES, Institute of Crop Science and Resource Conservation, Crop Bioinformatics, University of Bonn, 53115 Bonn, Germany
5IZMB, Institute of Cellular and Molecular Botany, Department of Ecophysiology, University of Bonn, 53115 Bonn, Germany

The monogenic recessive mutant dizzy1 (diz1) exhibits a dwarfed and twisted leaf and root phenotype. We identified diz1 in a phenotypic screen of an F2 population derived from the Mutator-tagged BonnMu resource. Histological analysis of diz1 mutants revealed abnormal cell organization in seedling leaves and roots. In leaves, upper epidermal cells were enlarged, while in roots the cortical cells showed an irregular arrangement. Physiological analysis of primary roots indicated reduced cell viability in diz1. This was reflected by increased membrane permeability and altered metabolic activity. To identify DIZ1-dependent gene expression patterns, we performed comparative RNA-seq on primary roots of diz1 mutants and the wild-type. In total, 4,378 genes were differentially expressed. Functional enrichment analysis of the differentially expressed genes pointed to elevated reactive oxygen species and lignin accumulation in diz1 roots, which we confirmed through quantitative measurements. Moreover, diz1 mutants were insensitive to exogenous brassinolide application and showed delayed shoot promotion by 1-naphthaleneacetic acid. The mutants also displayed strong inhibition of lateral root formation in response to gibberellin A₃. A bulked segregant RNA-sequencing analysis mapped the candidate gene underlying the diz1 phenotype to chromosome 2. Prioritization of the most likely candidate gene is currently underway.

P114: Development of efficient haploid inducers of fast flowering mini maize

Cell and Developmental Biology James Birchler (Principal Investigator)

Zhang, Zhengzhi1
Liu, Hua1
Yang, Hua2
Gao, Zhi2
Albert, Patrice S.2
Char, Si Nan1
Birchler, James A.2
Yang, Bing1

1Division of Plant Science and Technology, University of Missouri, Columbia, MO 65211
2Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Doubled haploid technology enables the rapid generation of completely homozygous lines in a single generation, thereby accelerating the development of true-breeding germplasm in crop breeding. In maize, haploid inducer (HI) lines can be generated by modifying the PLA1/NLD/MTL and DMP genes, resulting in the production of maternal haploids when the HI is used as the pollen donor. In the present study, Fast Flowering Mini Maize (FFMM) was used for CRISPR-Cas9 based gene-editing of the ZmMTL and ZmDMP genes. To facilitate haploid identification, GFP and RUBY markers were incorporated into the edited lines, generating HI lines with different combinations of haploid induction alleles. Progeny from crosses between Mo17 and these HI lines were initially screened for haploids based on the absence of GFP fluorescence in seedling roots. HI lines carrying only the edited mtl allele (mtle) or the stock6 mtl allele (mtls6) produced haploids at rates of 3.3% and 6.3%, respectively, whereas editing of ZmDMP alone resulted in a haploid induction rate of 1.2%. In contrast, HI lines carrying both mtle and the edited ZmDMP allele (mtle/dmp) or mtls6 and ZmDMP edited allele (mtls6/dmp) exhibited significantly higher haploid induction rates of 15.0% and 14.0%, respectively. Haploid plants treated with nitrous oxide gas (600 kPa for 2 days) at the six-leaf stage developed fertile tassel sectors. Notably, untreated haploids were also able to produce normal kernels when pollinated with normal pollen, likely due to meiotic failure or spontaneous genome doubling during ear development. The mtle/dmp HI line was further evaluated for haploid induction in Mexican teosinte (Zea mexicana), achieving an induction rate of 18.8%, comparable to that observed in maize. Owing to its compact stature and flowering time of 30 days or less under standard greenhouse conditions, Fast Flowering Mini Maize provides an efficient platform for haploid induction in maize and related species. @font-face {font-family:“Cambria Math”; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:“Arial Unicode MS”; panose-1:2 11 6 4 2 2 2 2 2 4; mso-font-charset:128; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-134238209 -371195905 63 0 4129279 0;}@font-face {font-family:“Liberation Serif”; panose-1:2 11 6 4 2 2 2 2 2 4; mso-font-alt:“Times New Roman”; mso-font-charset:1; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:0 0 0 0 0 0;}@font-face {font-family:“Songti SC”; panose-1:2 1 6 0 4 1 1 1 1 1; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:647 135200768 16 0 262303 0;}@font-face {font-family:“@Songti SC”; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:647 135200768 16 0 262303 0;}@font-face {font-family:“@Arial Unicode MS”; panose-1:2 11 6 4 2 2 2 2 2 4; mso-font-charset:128; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-134238209 -371195905 63 0 4129279 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:““; margin:0in; mso-pagination:widow-orphan; mso-hyphenate:none; font-size:12.0pt; font-family:”Liberation Serif”,serif; mso-fareast-font-family:“Songti SC”; mso-bidi-font-family:“Arial Unicode MS”; mso-font-kerning:1.0pt; mso-fareast-language:ZH-CN; mso-bidi-language:HI;}span.StrongEmphasis {mso-style-name:“Strong Emphasis”; mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:““; font-weight:bold;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:”Liberation Serif”,serif; mso-ascii-font-family:“Liberation Serif”; mso-fareast-font-family:“Songti SC”; mso-hansi-font-family:“Liberation Serif”; mso-bidi-font-family:“Arial Unicode MS”; mso-ligatures:none; mso-fareast-language:ZH-CN; mso-bidi-language:HI;}.MsoPapDefault {mso-style-type:export-only; mso-hyphenate:none;}div.WordSection1 {page:WordSection1;}

P115: Developmental and environmental control of genetic interactions between GRFs and TCPs.

Cell and Developmental Biology Stijn Seynnaeve (Graduate Student)

Seynnaeve, Stijn1 2
Beauchet, Arthur1 2
Lorenzo, Christian D1 2
Wytynck, Pieter1 2
Blasco-Escámez, David1 2
Sanches, Matilde1 2
Vandeputte, Wout1 2
Nelissen, Hilde1 2

1Center for Plant Systems Biology, VIB, B-9052 Gent, Belgium
2Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, Belgium

A major challenge in crop improvement is translating gene function insights obtained under controlled conditions into robust performance gains in the field. In maize, quantitative growth traits are governed by complex regulatory gene networks whose developmental and environmental context dependence often limits their predictive value outside the laboratory. Previous multiplex genome editing identified members of the GROWTH REGULATING FACTOR (GRF) and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor families as key regulators of seedling leaf growth. Here, we systematically dissect GRF–TCP genetic interactions across developmental stages and environments to evaluate their translational relevance from controlled settings to field conditions. Using defined single and double mutations in GRF and TCP genes, we identified both additive and non-additive interactions affecting seedling leaf growth. Notably, the grf10 tcp42 double mutant exhibited a synergistic increase in leaf length relative to wild type and the corresponding single mutants, whereas other combinations showed antagonistic effects, revealing strong context dependence within this regulatory network. In addition, phenotyping grf10 tcp42 at later developmental stages and under field conditions confirmed the developmental and environmental control of the phenotypic outcome. Together, these results highlight the lab-to-field translational gap in growth-related gene interactions and ongoing comparative transcriptomic analyses under controlled and field conditions will further elucidate the molecular basis of environment-dependent phenotypic penetrance. This work provides a framework for improving the translational deployment of gene interaction knowledge toward agronomically relevant trait development in maize through plant biotechnology.

P116: Difference in variation in root anatomy along the longitudinal axis between maize and temperate cereal crops

Cell and Developmental Biology Maria Schön (Graduate Student)

Schön, Maria1
Jones, Dylan H.1
Schneider, Hannah M.1

1Leibniz Institute of Plant Genetics & Crop Plant Research (IPK) OT Gatersleben, Corrensstr 3, 06466 Seeland, Germany

Root system architecture and anatomy are key determinants of plant performance under stress, yet our understanding of root anatomical plasticity remains limited due to spatially restricted sampling strategies. In particular, the lack of systematic analyses along the entire root axis hampers insights into how anatomical traits contribute to resource-use efficiency and yield stability. This study investigates changes in vascular anatomy along the root axis of nodal roots in four temperate crop species—Triticum aestivum (wheat), Secale cereale (rye), Hordeum vulgare (barley) — the grain cereal Avena sativa (oat) as well as one tropical species, Zea mays (maize). Whole root systems were sampled intact from soil grown plants and root vascular anatomical traits were quantified at multiple positions along the root axis of different root classes. In addition, root architecture, with a particular focus on lateral root formation and root growth rate, was investigated in wheat alongside the anatomical study. These surveys reveal a range of changes in vascular patterning along the root axis. A reduction in metaxylem number along the longitudinal root axis was observed in all species, although this occurred to different extents when comparing maize to temperate cereals. Furthermore, changes in metaxylem size varied among species. In wheat, changes in vascular anatomy were not associated with distinct patterns of lateral root formation and root growth dynamics. Together, these results demonstrate that there is much to be explored in the links between root anatomy and root architecture to provide new insights relevant for crop improvement.

P117: Disrupted gamete interaction in maize gex2 mutants biases fertilization toward egg-cell-only fertilization and promotes heterofertilization

Cell and Developmental Biology Andrea Calhau (Graduate Student)

Calhau, Andrea R.M.1
Flieg, Harrison2
Duvernois-Berthet, Evelyne1
Vejlupkova, Zuzana2
Fowler, John2
Widiez, Thomas1

1Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon1, CNRS, INRAE, Lyon, France.
2Oregon State University; United States; Corvallis, Oregon, 97331

The pollen grain delivers two sperm cells to the female embryo sac to accomplish double fertilization, a process involving two coordinated and distinct fusion events that initiate seed development. Although this process is fundamental for kernel formation, the molecular mechanisms that ensure male–female gamete interactions remain incompletely understood. Building on the identification of the sperm cell surface protein GAMETE EXPRESSED 2 (GEX2), which is required for gamete attachment in Arabidopsis, we investigated its function in maize using two gex2 mutant alleles that display reduced male transmission and elevated kernel abortion. By combining a fluorescent sperm cell marker line with a newly developed imaging method of the embryo sac, we gained unprecedented access to the earliest cellular events of double fertilization in maize, enabling assessment of the effects of the most severe gex2 allele. When gex2 mutant pollen was used, approximately 70% of embryo sacs contained unfused sperm cells, compared with ~8% in wild-type controls, indicating a severe defect in male-female gamete interaction. Notably, gex2 sperm cells resulted in elevated egg-cell single fertilization. Analysis of embryo and endosperm parentage in mature kernels from gex2 pollinations using an anthocyanin marker revealed a significantly increased rate of heterofertilization, in which the egg cell and central cell are fertilized by different pollen tubes. Consistent with the observed bias toward egg-cell fertilization, we found that ~90% of the heterofertilized kernels were associated with an embryo originating from gex2 fertilization. RNA-seq analysis reveals that the differing phenotypic severity of the two gex2 mutant alleles, as measured by transmission rate, likely arises from the production of distinct truncated GEX2 proteins. Collectively, these data provide cellular and molecular evidence supporting a critical role for GEX2-dependent male–female gamete recognition in ensuring proper and balanced double fertilization in maize.

P118: Dissecting the gene regulatory networks controlling maize nucellus degeneration

Cell and Developmental Biology Jie Zhang (Postdoc)

Zhang, Jie1 2
Fontanet-Manzaneque, Juan B.1 2
Sun, Geng1 2
Lee, Jimin1 2
Aesaert, Stijn1 2 3
Coussens, Griet1 2 3
Hoengenaert, Lennart1 2 3
De Rycke, Riet4
Doll, Nicolas M.5
Nowack, Moritz K.1 2

1Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
2Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.
3Crop Genome Engineering Facility, VIB, 9052 Ghent, Belgium.
4Bioimaging Core, VIB, 9052 Ghent, Belgium.
5Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, CNRS, INRAE, F-69342, Lyon, France.

The nucellus is a transient maternal tissue present in maize (Zea mays L.) kernels prior to fertilization. Following fertilization, the nucellus undergoes rapid expansion that contributes to early kernel growth, after which it is swiftly eliminated, creating space and releasing nutrients for the developing endosperm. Although programmed cell death (PCD) has been implicated in nucellus elimination, the gene regulatory networks controlling this process remain poorly defined. Using published RNA-seq datasets, we identified a set of nucellus-preferred transcription factors (TFs) that are strongly upregulated during nucellus degeneration. Functional screening in heterologous systems—including Nicotiana benthamiana agroinfiltration and maize leaf protoplast transfection—revealed several TFs capable of inducing cell death and activating PCD-associated signature genes. To assess TF function in vivo, dominant-negative versions of selected candidates were overexpressed specifically in the nucellus. Overexpression of a dominant-negative allele of one candidate, which we term Nucellus Elimination1 (Nel1), significantly delayed nucellus degeneration. This delay was associated with reduced endosperm size, indicating that timely nucellus elimination is required for optimal early kernel development. Ongoing work aims to identify additional components of the nucellus PCD regulatory network and to determine whether signals from the developing endosperm contribute to activation of this pathway. Elucidating the mechanisms governing nucellus elimination will advance our understanding of maternal–filial interactions and may reveal new targets for improving kernel development and yield in maize.

P119: Dissecting the boron deficiency response of maize roots using phenylboronic acid

Cell and Developmental Biology Liuyang Chu (Graduate Student)

Chu, Liuyang1
Matthes, Michaela1

1INRES-Crop Functional Genomics, University of Bonn, Römertsr. 164, Bonn, NRW, Germany, 53117

The micronutrient boron contributes to the stability of the primary cell wall by crosslinking two rhamnogalacturonan II (RG-II) subdomains of pectin, thereby forming RG-II dimers. Consequently, boron deficiency causes a reduction of RG-II dimerization in the cell wall. Moreover, boron deficiency leads to drastic alterations of phytohormone levels in the Arabidopsis roots, including a well-documented increase in auxin levels. Therefore, additional functions of boron revolving around hormone-mediated pathways were proposed. Whereas boron-hormone interactions have been identified during maize shoot development, the impact of boron deficiency on maize root development (including potential boron-hormone interactions) remains elusive. We, therefore, made use of phenylboronic acid (PBA), a boric acid analog, to systematically characterize maize primary root development under boron-deficient conditions. PBA was suggested as a tool to mimic boron deficiency in plants due to a putative interference with RG-II dimerization. Germination assays with PBA resulted in maize seedlings with impaired primary root elongation and a severe reduction in lateral root density compared to control treatments, resembling reported boron deficiency-induced phenotypes. Moreover, both PBA and boron-free treatments reduced auxin levels and increased levels of reactive oxygen species (ROS) in primary roots. These results highlight similarities of PBA-induced defects with boron deficiency-induced defects on the molecular level and exemplify striking differences in hormonal regulations under boron deficiency in maize compared to particularly Arabidopsis. Most notably, PBA did not interfere with RG-II crosslinking in the cell wall and maize root phenotypes induced by boron-free treatments were marginal. Our study, therefore, revealed unique phenotypic and molecular responses of maize roots under boron deficiency and particularly highlights crosstalk of boron and PBA with auxin and ROS. This crosstalk, which appears instrumental for the mode of action of PBA, opens new avenues for decoding lateral root development in maize and to elucidate novel boron functions in plants.

P120: ESR-localized CLE peptides regulate maize embryonic symmetric patterning

Cell and Developmental Biology Meng Hu (Graduate Student)

Hu, Meng1
Widiez, Thomas2
Dresselhaus, Thomas1
Jiang, Guojing1

1Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg; Universitätsstraße 31; Regensburg, Bavaria, Germany D-93053
2Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE; 46 Allée d'Italie; Lyon, Auvergne-Rhône-Alpes, France F-69342

The endosperm is a transient nutritive tissue in plant seeds, but in maize it also harbors the Embryo Surrounding Region (ESR), a specialized cell layer hypothesized to act as a physiological buffer and signaling center, modulating the hormonal environment and communication with the developing embryo. We selected three ESR-specific genes for functional studies, ESR1, ESR2 and ESR3, encoding CLE family peptides, to elucidate the role of the ESR. Using a pESR2::GFP reporter line, we found that ESR2 is specifically expressed in the cells around the suspensor of embryos at 7 days after pollination (DAP), when rapid embryo growth breaks through the ESR layer, allowing the embryo to directly perceive developmental signals from the endosperm, which coincides with the onset of bilateral symmetry and establishment of the shoot–root axis. CRISPR/Cas9-generated triple esr1,2,3 mutants exhibit shifted orientation and repositioning of the shoot–root axis, indicative of disrupted embryonic symmetry patterning. Ongoing work using ESR2-driven cell death inducer KIL1 to ablate ESR cells will directly test the necessity of this cell layer for embryogenesis. Together, our data indicate that ESR-derived CLE peptides play a crucial role in controlling embryonic patterning in maize. These findings provide insights into cereal embryo development and may inform strategies for crop improvement.

P121: Early patterning and morphogenesis of the maize embryo

Cell and Developmental Biology Mingchen He (Graduate Student)

He, Mingchen1
Dresselhaus, Thomas1
Jiang, Guojing1

1Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, 93053 Regensburg, Germany

Maize (Zea mays L.) embryos exhibit a specialized developmental architecture that differs markedly from that of the dicot model Arabidopsis thaliana. During maize embryogenesis, a single cotyledon (known as scutellum), a coleoptile enclosing the embryonic shoot, successive embryonic leaves, and a highly regionalized plumule–radicle axis are formed, giving rise to a structurally complex embryo typical of grasses. In this study, we provide a refined description of the early developmental trajectory of the maize embryo. We show that the shift from radial to bilateral symmetry is initiated during the early- to mid-transition stage. Between the mid- and late-transition stages, the shoot and root apical meristems, together with the coleoptile, become morphologically defined. The first embryonic leaf primordium, marked by DRL1 (Drooping Leaf1) expression, emerges by the late transition stage in the region below the shoot apical meristem (SAM), indicating that the SAM is already competent for organ differentiation at this time. At leaf stage 1, embryonic morphogenesis is largely completed. These key morphological transitions coincide with significant shifts in transcriptional programs. Together, our findings indicate that rapid and precise morphogenesis of the maize embryo is likely driven by highly coordinated and complex gene regulatory networks.Keywords:Maize, Embryogenesis, Leaf primordium, Shoot apical meristem

P122: Effector-driven developmental reprogramming during Ustilago maydis–induced gall formation

Cell and Developmental Biology Mamoona Khan (Postdoc)

Khan, Mamoona1

1Department of Plant Pathology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn 53113, Germany

The corn smut fungus Ustilago maydis induces gall formation on all aerial parts of the maize plant. Gall formation requires profound developmental reprogramming, yet the host pathways that enable this plasticity remain poorly defined. We utilized fungal effectors as targeted developmental perturbations to identify plant pathways capable of driving gall-like growth programs. By expressing individual U. maydis effectors in Arabidopsis thaliana, we uncovered fundamentally distinct developmental pathways that can induce proliferative, gall-like phenotypes. These pathways engage different regulatory modules and revealed that pathogen-driven growth can arise through multiple, mechanistically independent routes. One of the identified pathways operated through effector-mediated interference of the highly conserved TOPLESS/TOPLESS-RELATED family of transcriptional corepressors, leading to de-repression of an auxin-responsive transcriptional program associated with lateral root initiation. Effector expression induced pluripotent, callus-like growth in A. thaliana and activated developmental gene networks that strongly overlapped with lateral root formation in the host plant, Zea mays. Genetic analyses identified LATERAL ORGAN BOUNDARIES DOMAIN regulators as essential nodes in this response, and maize homologs of these genes modulated gall development in planta, linking effector activity to conserved developmental control mechanisms.

P123: Embryo-derived signaling regulates nutrient allocation in maize kernels

Cell and Developmental Biology Laurine Grazer (Graduate Student)

GRAZER, Laurine1
LEMAIRE, Edgard1
MONTES, Emilie1
GOMEZ, Victoria2
INGRAM, Gwyneth1
WIDIEZ, Thomas1

1Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France
2Institut de Biologie Paris-Seine - IBPS - Sorbonne Université, IBPS, Sorbonne Université, CNRS, Paris, France

Maize kernels contain three compartments: the embryo, the endosperm, and the pericarp which can be likened to a family. As in any family, a balance exists between competition, mutual aid, and self- sacrifice for space and nutrients. In addition, Kernels also compete with one another, underlying a major unanswered question: Can the mother plant preferentially allocate nutrients to kernels that are more likely to survive? To address this question, we compared segregating kernels with and without embryos from heterozygous emb mutant plants. These kernels show pronounced differences in weight and morphology, leading us to question the role of the embryo in kernel development and the possibility of an embryo-driven signaling pathway influencing maternal nutrient allocation. The team identified an interesting candidate gene, whose expression in the endosperm strictly depends on the presence of the embryo. This gene encodes a protein belonging to the PEBP (Phosphatidylethanolamine Binding Protein) family, its members show symplastic mobility and modulate transcription factor and nutrient transporter activity. Our PEBP of interest belongs to the MFT, (Mother of FT and TFL-1) subfamily, and is named pebp11. To test its role, we generated and characterized CRISPR KO mutants and ectopic expression lines in terms of kernel nutrient content, morphology and metabolites. Our preliminary results show that pebp11 mutants have reduced lipid content in the embryo. Suggesting that PEBP11 may tune embryo nutrition. Aware of redundancy in maize we generated pebp9;10;11 mutants, which germinate before drying. We showed that this is due to loss of function of pebp9 and pebp10, which are expressed in the embryo. To obtain mechanistic insights, we examined the mobility and interactions of PEBP11, revealing further clues about the molecular basis of its function. Overall, our results support a model in which embryo-derived signals may modulate endosperm function and maternal nutrient supply during kernel development.

P124: Exploring the spatial dynamics of root cortical barriers in maize stress responses

Cell and Developmental Biology Darshan Damodaran (Graduate Student)

Damodaran, Darshan1
Schneider, Hannah1

1Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany, 06466

Root cortical barriers are considered potential game changers for improving stress resilience under adverse soil conditions such as drought and compaction. Maize, one of the most widely cultivated cereal crops with major global economic importance, is increasingly exposed to unfavourable soil environments due to unpredictable climatic conditions and soil degradation. These factors collectively place significant stress on maize root systems, ultimately reducing yield. In this study, we investigated cortical cell barriers in maize roots, focusing on the endodermis, exodermis, and multiseriate cortical sclerenchyma. These barrier forming tissues originate from the root cortex and differentiate into specialized cell types as the plant grows, adopting distinct functional roles. Using root anatomical and architectural phenotyping, we examined the development of these barriers along the entire root length under greenhouse conditions in drought and compaction stress. Our findings show that the development of root cortical barriers varies along the root axis under different stress conditions and also differs among root types. This suggests that maize roots respond to stress by initiating earlier and more substantial barrier deposition to withstand adverse conditions. Understanding the spatial and temporal variation in cortical barrier formation under stress is therefore critical for developing maize varieties with improved resilience and enhanced yield.

P125: Genome editing efficiency during haploid induction is maximized through cenh3-mediated genome elimination

Cell and Developmental Biology Fisher Stines (Research Scientist)

Stines, Fisher1
Zhang, Xia1
Dinwiddie, Jay1
Kershner, Karleigh1
McNamara, Dawn1
Zhu, Ling1
Carter, Jared1
Liang, Dawei2
Geng, Lizhao2
Kelliher, Timothy3
Egger, Rachel1

1Seeds Research & Development, Syngenta, Research Triangle Park, North Carolina, USA 27709
2Syngenta Biotechnology China Co., Beijing, China
3Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, USA 27695

Haploid Induction (HI) coupled with CRISPR-Cas12a genome editing provides a transformative tool for direct genome editing in maize, yet the narrow temporal window for nuclease activity between double fertilization and uniparental chromosome elimination limits editing efficiencies and inhibits widespread adoption of the technology. HI-Edit CenH3 leverages the longer temporal window for nuclease activity with cenh3-mediated genome elimination to enhance editing efficiency. Initial characterization of this system achieved a Haploid Editing Rate (HER) of 17% across four elite inbred tester lines. To further enhance HER, we developed a heat treatment and integrated an engineered Mb2Cas12a nuclease to achieve a combined five-fold increase in HER targeting multiplexed genic and intergenic loci, with examples reaching 100% efficiency. This enhanced system demonstrated robust performance and broad applicability, achieving high HER across more than 30 diverse elite inbred lines. Paternal cenh3-mediated HI-Edit offers a highly efficient, broadly applicable, and streamlined method for precise genome editing in maize, circumventing traditional breeding bottlenecks and accelerating crop improvement.

P126: Grafting in maize

Cell and Developmental Biology Shijun Zhang (Graduate Student)

Zhang, Shijun1
Zimmerman, David2
Kragler, Friedrich1

1Max Planck Institut für Molekulare Pflanzenphysiologie, Wissenschaftspark Golm, Am Mühlenberg 1, 14476 Golm, Germany
2School of Biological Sciences, Cold Spring Harbor Laboratory, 1 Bungtown Rd, Cold Spring Harbor, 11724 NY, United States

Transport of signalling macromolecules between tissues is one of the basic mechanisms coordinating the development and growth of higher plants. Local or long-distance exchange of RNAs and proteins between tissues facilitates plant adaptation to stress conditions, guides flowering time and apical growth and development. Heterografting has served as a key method for tracing signalling macromolecules exchanged between different plant parts. While grafting and multi-omics approaches have provided a wealth of information on mobile macromolecules of dicots, we have very limited information on the macromolecules moving in monocots, particularly maize. The identification of graft-mobile macromolecular signals in maize has been hindered by the lack of a robust hetero-grafting protocol suitable to allow long-term growth of plants. To address this, we test and try to implement several variant grafting protocols for maize. The grafting approaches and preliminary results will be presented. Keywords: grafting, maize, macromolecular transport

P127: Heat stress at tricellular stage hinders maize pollen dehiscence

Cell and Developmental Biology Divya Rana (Graduate Student)

Rana, Divya1
Kim, Taehoon1
Begcy, Kevin1

1University of Florida, Microbiology and Cell Science department, Gainesville, Florida, USA, 32611

Global warming has caused a significant increase in the frequency and intensity of heatwaves and hot days worldwide. Exposure to high temperatures during the plant reproductive phase leads to dramatic decrease in seed production. Moreover, male reproductive organs including anthers and pollen are more sensitive to temperature fluctuations than female gametophytes. While the impact of heat stress at the tetrad, unicellular and bi-cellular stage of pollen development has been elucidated, it is not known how heat stress (HS) impacts the tricellular stage of pollen development. Using three maize (Zea mays) inbred lines (B73, W22 and A188), we imposed a moderate heat stress (35°C/25°C light/dark period) for 48 h on maize plants, specifically at the tricellular stage. We found that HS at the tricellular stage resulted in reduced starch content, decreased enzymatic activity, and reduced in vitro and in vivo pollen germination rates. In addition, HS delayed the anther dehiscence and significantly reduced the seed set of all three inbred lines. Transverse sectioning of heat stressed anthers demonstrate that heat stress delays the disintegration of endothecium layer, while non-stressed plants complete disintegration of the endothecium layer in anthers and thus leading to reduced pollen release. To understand the molecular basis of these phenotypes, we performed RNA-Seq analysis on anthers and pollen grains under HS and control conditions. Among the differentially expressed genes, HS yielded MADS2. This transcription factor is required for anther and pollen maturation in maize and accumulates in apoptotic bodies during anther dehiscence. Using transgenic plants carrying the ZmMADS2-green fluorescent protein (GFP) fusion protein under control of the ZmMADS2 promoter, we performed Co-immunoprecipitation and ChIP-Seq assays to identify interactors and downstream genes of this gene.

P128: High-resolution, stage-specific single-cell atlas reveals meristem transitions in maize inflorescence development

Cell and Developmental Biology Xiaosa Xu (Principal Investigator)

Xu, Xiaosa1 2
Beverly, Sorickya1
Yang, Ruoming1
Wang, Zhen1
Wang, Wendy1
Passalacqua, Michael2
Li, Rachel1
Gillis, Jesse2 3
Jackson, David1

1Department of Plant Biology, University of California, Davis, CA 95616, USA
2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
3Department of Physiology, University of Toronto, Toronto, Ontario, M5S 3E1, CANADA

Maize productivity relies on the precise regulation of inflorescence development, which entails coordinated meristem fate transitions and complex communication among diverse cell populations. However, elucidating these processes has been hindered by genetic redundancy, pleiotropy, and the morphological complexity of maize meristems. Our previous single-cell RNA sequencing (scRNA-seq) of whole ear primordia provided an initial overview of cell types and meristem domains but lacked the resolution to resolve dynamic transitions between meristem states. To overcome this limitation, we generated a high-resolution, stage-specific scRNA-seq atlas of developing maize ears by precisely dissecting four key meristematic stages: the inflorescence meristem/early spikelet pair meristem (IM/SPM), late spikelet pair meristem (SPM), spikelet meristem (SM), and floral meristem (FM). Cluster annotation identified conserved cell types and spatial domains across developmental stages, while differential expression analysis revealed dynamic transcriptional programs underlying meristem fate transitions. In parallel, we constructed a high-resolution scRNA-seq atlas of developing tassel inflorescences. Tassels, unlike ears, form branches, and through fine dissection and single-cell profiling of tassel branch primordia, we found that their cell type composition closely resembles that of maize ear tips. This provides valuable insights into conserved and divergent cellular programs underlying the architectural differences between tassels and ears. Finally, we identified candidate genes with dynamic expression across meristem stages and employed CRISPR-based functional analysis to characterize their roles. This work establishes the most comprehensive single-cell gene expression resource for maize inflorescence development to date, providing critical insights into the regulatory logic of meristem transitions and a foundation for strategies to improve grain yield and plant architecture.

P129: How to reproducibly pattern a meristem despite size variability

Cell and Developmental Biology Cecilia Lara-Mondragón (Postdoc)

Lara-Mondragón, Cecilia1
Feller, Antje1
Knauer, Steffen1
Timmermans, Marja1

1Center for Molecular Biology of Plants (ZMBP), University of Tübingen, Tübingen, Germany, 72076

Maize domestication and selective breeding resulted in considerable morphodiversity, observable through the vegetative and reproductive phases across breeding stocks. At the microscopic level, such diversity is seen in the shape and size of the vegetative Shoot Apical Meristem (SAM), and this variation offers a unique system within which to investigate the effect of size in organ patterning. SAM morphodiversity was explored in 3D, revealing evidence for proportional pattern size-adjustment (scaling) of the SAM functional domains across inbred lines. To arrive at potential mechanism(s) underlying pattern scaling in the SAM, size measurements and RNA-seq were integrated to fit a linear model. This analysis identified a subset of genes with strong association to SAM volume. Among these candidates, the expression of known regulators of SAM homeostasis such as genes involved in cytokinin metabolism displayed opposite and complementary behavior as a function of SAM size, an observation corroborated in vitro. Our work suggests that SAM size regulation is a complex, layered process that may exhibit dose-dependent dynamics. Altogether, this work delves into a fundamental, yet largely unexplored question in plant developmental biology, namely how developmental patterns scale.

P130: Integrating replication timing, transcriptional activity, and 3D genome organization in maize

Cell and Developmental Biology Hank Bass (Principal Investigator)

Bass, Hank W.1
Akram, H. Sara1
Wear, Emily E.2
Mickelson-Young, Leigh2
Wheeler, Emily2
Concia, Lorenzo3
Thompson, William F.2
Hanley-Bowdoin, Linda K.2

1Department of Biological Science, Florida State University, Tallahassee, FL, USA
2Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA
3Texas Advanced Computing Center, University of Texas, Austin, TX, USA

Eukaryotic DNA replication is a highly organized process in which different genomic regions are duplicated in a defined order. Replication time (RT) is an inherent property of individual genome segments that integrates many aspects of genome structure and function, including transcriptional activity, chromatin structure, and 3D nuclear architecture. We have developed two approaches to study RT in the root tips of maize seedlings. Wheeler et al. (2025, PMID: 40404767) showed that RT profiles generated by Repli-Seq (sequencing of replicated DNA in nuclei at different stages of S phase) or by DNA copy number analysis (ratio of S/G1 DNA content) are very similar. Repli-Seq provides higher RT resolution and can resolve heterogeneity in a population of nuclei, while S/G1 requires less material and can be used for large numbers of samples. Wear et al. (2025, PMID 40795170) found that steady-state nuclear RNA is remarkably similar to newly synthesized nuclear RNA and both are distinct from cellular RNA. Both types of nuclear RNA data allow for efficient detection of low-abundance coding and non-coding RNAs, include a significant fraction of intronic reads, and are excellent indicators of transcriptional activity for comparison to RT data. Akram et al. (2025, BioRxiv) used a combination of Hi-C and 3D-FISH to show that maize sequences that replicate in early versus middle S phase represent distinct euchromatic sub-compartments. The early-S regions are more enriched for open, transcriptionally active chromatin and have more long-range contacts than middle-S regions. The early and middle-S regions are often intimately interspersed along maize chromosome arms but occupy largely non-overlapping nucleoplasmic regions throughout all stages of interphase, including G1. Our findings highlight the importance of replication timing as a conserved feature of maize chromatin architecture.

P131: Leaf-based transformation as a strategy for the genetic transformation of tropical maize lines

Cell and Developmental Biology Maria Helena Faustinoni Bruno (Graduate Student)

Bruno, Maria Helena F1 2
Pinto, Maísa S1
Nonato, Juliana1
Pauwels, Laurens3
Arruda, Paulo1 2
Yassitepe, Juliana1 4

1Genomics for Climate Change Research Center (GCCRC), Universidade Estadual de Campinas, Campinas, SP, Brazil,13083-875
2Departamento de Genética, Evolução, Microbiologia, e Imunologia- Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil, 13083- 970
3Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium
4Embrapa Agricultura Digital, Campinas, SP, Brazil, 13083-886

Maize genetic transformation is a key method for developing new varieties. Tropical genotypes often display high recalcitrance, leading to low transformation rates. The use of morphogenic genes (MRs), such as Baby Boom (Bbm) and Wuschel (Wus), improved transformation outcomes even for previously recalcitrant maize genotypes and enabled exploration of different explant types. Foliar tissues show promise, with positive results in maize, sorghum, and bean. We evaluated transforming maize leaf tissue from the B104 genotype and the tropical lines CML360 and CML488. We used a leaf-based protocol and Agrobacterium strains EHA105 and LBA4404, each with the ternary vector pVS1-VIR2 and a binary vector carrying an expression cassette with the NptII gene, an MR cassette with Wus2, a 3xEnh targeting Bbm, and the Cre recombinase under the Hsp26 promoter. With EHA105, we regenerated one plant each of B104 and CML488, but none of CML360. Although our group had never transformed the CML488 line using immature embryos, leaf tissue transformation allowed us to produce a transformed plant of this genotype for the first time. Using LBA4404, we regenerated 11 plants of CML360, one of CML488, and four of B104. All plants tested positive for NptII. The transformation efficiency for B104, CML360, and CML488 using EHA105 was, respectively, 4.16%, 0%, and 3,33%. With LBA4404, it was 16%, 44%, and 8,33%, respectively. To excise the morphogenic genes, we subjected plants to heat shock at 45 °C for 2 hours under 70% humidity. CML plants subjected to heat shock showed 100% excision of Wus2. For B104, only one plant excised Wus2. Our results demonstrate that the LBA4404 strain is promising for this type of transformation, and leaf tissue transformation is a viable and effective strategy for maize, including recalcitrant tropical genotypes.

P132: Linking carbon metabolism to meiotic entry in maize anthers: From bulk to single-cell resolution

Cell and Developmental Biology An Hsu (Graduate Student)

Hsu, An1
Cheng, Chia-Yi1
Wang, Chung-Ju Rachel2

1Department of Life Science, National Taiwan University, Taipei, 106319, Taiwan (R.O.C)
2Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 115201, Taiwan (R.O.C)

Anther development is pivotal for crop yield and plant breeding, involving coordinated cell division and differentiation that generate four specialized somatic layers enclosing germinal cells. Spatiotemporal interactions among these layers orchestrate germinal cell development from mitotic proliferation through meiosis and ultimately pollen formation. However, the mechanisms by which somatic layers coordinate and support the transition of germinal cells from mitosis to the meiotic program remain poorly understood. In this study,we analyzed published bulk RNA-seq and scRNA-seq datasets and identified significant upregulation of genes associated with carbon metabolism and energy production. Notably, the onset of this upregulation coincides with the transition to meiosis, rather than during the early stages of anther development characterized by extensive cell division. Supporting this finding, quantification of soluble sugars in whole anthers showed increased sucrose concentrations as anthers approached meiosis, suggesting a possible metabolic reprogramming involved in meiotic entry. Given that the anther is a sink organ for photosynthates, histological staining of transverse sections revealed abundant starch accumulation in the endothecium before meiosis begins, indicating its role as a local energy reserve. These observations suggest that carbohydrate dynamics in the anther may provide carbon sources or signaling cues to germinal cells. To further explore regulatory mechanisms, we constructed gene regulatory networks (GRNs) for meiocytes and whole anthers. The meiocyte single-cell GRN revealed putative metabolic pathway–associated regulons specifically active during the transition phase. In the whole-anther GRN, the putative transcription factors previously reported to be involved in anther meiotic progression, were highlighted as candidates regulators. Together, we propose that elevated energy metabolic activity may contribute to crosstalk and signaling between somatic niche cells and germinal cells during the mitosis-to-meiosis transition.

P133: Maize BABY BOOM transcription factors initiate parthenogenesis and embryonic patterning

Cell and Developmental Biology Xixi Zheng (Postdoc)

Zheng, Xixi1
Tornero, Maria Flores1
Schwartz, Uwe2
Dresselhaus, Thomas1

1Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, 93053 Regensburg, Germany
2Computational Core Unit, University of Regensburg, 93053 Regensburg, Germany

Parthenogenesis, in which an embryo arises from an unfertilized egg cell, is central to seed-based asexual reproduction known as apomixis. We found that parthenogenesis occurs in all investigated polyploid lines of Tripsacum dactyloides, the closest wild relative of maize. A comparison of sexual and parthenogenetic egg cell transcriptomes of T. dactyloidesrevealed that egg cell activated expression of the AP2-EREBP family transcription factor BABY BOOM (BBM) in parthenogenetic egg cells largely accounts for this process. By using a transgenic reporter line in maize, we show that BBM accumulates in nuclei of embryonic shoot–root axis cells, the developing provascular system, and the coleoptile and leaf primordia, marking the onset of embryonic organogenesis. Consistently, higher-order bbm knockout mutants exhibit severe defects in shoot–root axis development during both embryogenesis and germination. Integrative analyses including transcriptomics and immunofluorescence suggest that impaired gibberellin (GA) signaling contributes to the phenotypic abnormalities observed in bbm mutants. bbm embryos exhibit defective shoot meristem formation and leaf initiation, as well as dysregulated hormone pathways. Next, we aim to elucidate how BBM initiates parthenogenesis and expands its regulatory role in embryonic patterning, particularly the embryonic leaf formation characteristic of monocots, which appears different to the post-embryonic roles of the homologs in the model dicot Arabidopsis.

P134: Maize development under boron-deficient field conditions: Is the benzoxazinoid pathway a target for improvement?

Cell and Developmental Biology Cay Christin Schaefer (Graduate Student)

Schäfer, Cay C1
Plotecki, Felix1
Best, Norman B2
Matthes, Michaela S1

1University of Bonn, INRES - Crop Functional Genomics; Römerstraße 164, 53117 Bonn, Germany
2USDA-ARS-MWA-PGRU, 203 Curtis Hall, Columbia, MO 65211, USA

Deficiency of the micronutrient boron causes severe developmental defects and yield losses in various crops, including maize (Zea mays L.). So far, the only characterized molecular regulators of boron homeostasis in plants are boron transporters, it is hypothesized that additional factors contribute to regulating plant boron levels. To identify such factors in maize, a genome-wide association study based on natural variation in leaf boron concentration was performed. Most notably, this analysis detected benzoxazinless3 (bx3) and bx4, two genes of the well-characterized benzoxazinoid biosynthesis pathway, with its end product 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA). BX3 converts the pathway intermediate indolin-2-one (ION) to 3’-hydroxy-ION (HION), and BX4 converts HION to 2-hydroxy-1,4-benzoxazin-3-one. Strikingly, the bx3 mutant, which accumulates ION, showed increased leaf boron concentrations and enhanced tassel traits compared to B73 under low boron conditions. Therefore, bx3 mutants were systematically characterized across three field sites differing in soil boron levels (ranging from borderline boron-deficient to adequate). Vegetative traits were reduced or unchanged in bx3 mutants compared to B73 across all field sites, while particularly tassel branch number appeared enhanced in bx3 mutants under borderline boron-deficient conditions. A bx4 mutant is currently generated using the CRISPR/Cas9 technology. Furthermore, correlation analyses of bx3 and bx4 gene expression with boron concentration were performed in ear leaves of the Nested Association Mapping panel grown under low boron conditions in the field. Our study highlights novel molecular regulators of boron homeostasis and aids in resolving whether the benzoxazinoid biosynthesis pathway is a target for improving maize development in boron-deficient conditions.

P135: Modulation of host gene expression via fungal transactivation effectors (TAEs)

Cell and Developmental Biology Weiliang Zuo (Postdoc)

Zuo, Weiliang1
Xiao, Muye1
Doehlemann, Gunther1

1Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS); University of Cologne; Cologne, 50674, Germany

Phytopathogens secrete effectors that suppress plant immunity and modulate the host’s transcriptome for their infection. In a previous study, we identified Ustilago maydis effector Sts2 (small tumor on seedlings2), which functions as a transcriptional coactivator to activate the expression of maize leaf developmental regulators, resulting in the formation of hyperplastic tumors. Interestingly, the ortholog of Sts2 (SrSts2) from Sporisorium reilianum, the closest pathogenic relative of U. maydis, exhibits a diverse virulence function.We conducted a comprehensive evaluation of the transcriptional activation activities of all Sts2 orthologs identified in smut fungi, along with their computed ancestor, and tested their virulence function. Our results suggest that Sts2 orthologs represent a functional TAE (Transactivation effectors) family in smut fungi. During speciation, UmSts2 adapted its function to promote the specific tumor formation that is characteristic of U. maydis. Furthermore, AlphaFold analysis revealed two structurally variable domains between UmSts2 and SrSts2. Complementation assays using chimeric mutants carrying swapped variable domains validated that these domains determine the functional divergence of Sts2 in U. maydis and S. reilianum. In addition, we discovered several novel functional TAEs from two hemibiotrophic fungal pathogens, Magnaporthe oryzae and Bipolaris maydis, which infect rice and maize respectively. Heterologous expression of these TAE candidates in U. maydis reduced its virulence, indicating that they are virulence factors. Interestingly, overexpression of MoTAE from M. oryzae completely abolished U. maydis infection, likely due to its ability to induce host genes that regulate cell death in infected tissues. In summary, our results suggest that TAEs play a significant role in the pathogenesis of fungal infections.

P136: Molecular mechanisms modulating growth suppression in maize

Cell and Developmental Biology Jazmin Abraham-Juarez (Postdoc)

Abraham-Juarez, Jazmin1
Wang, Ludi1
Bartlett, Madelaine1

1Sainsbury Laboratory University of Cambridge, 47 Bateman Street Cambridge UK CB2 1LR

Plants have the plasticity to program and reprogram organ formation throughout their life cycle. Maize is an excellent model to study programming in organ formation. We discovered that the maize transcription factor GRASSY TILLERS 1 (GT1) and the trehalose-6-phosphate phosphatase RAMOSA3 (RA3) mediate both bud dormancy in axillary meristems and programmed cell death in pistils, which are key traits in plant architecture. gt1ra3 mutants show disruption of carpel suppression resulting in bisexual tassel flowers, and axillary bud suppression is disrupted resulting in a tillering phenotype. We found a synergistic genetic interaction between gt1 and ra3, with a stronger phenotype in the double mutant compared to the singles. However, it is unknown what molecular pathways modulate organ suppression to give different phenotypic outcomes (i.e. bud dormancy vs. pistil programmed cell death). Our aim is to use gt1 and ra3 as tool to study growth suppression at a molecular level in maize using different approaches. Specific antibodies against GT1 and RA3 are being used for protein localization in situ and immunoprecipitation followed by mass spectrometry. Also, interaction between GT1 and RA3 is being investigated. Our central hypothesis is that differential formation of protein complexes containing GT1 and RA3 drives differential transcriptional and developmental outcomes in flowers vs axillary buds. Studying the function of these proteins will elucidate the molecular basis of developmental reprogramming dependent on context, with a potential impact on organ identity modulation in grasses.

P137: Multiplex RCA FISH facilitates to visualize cell-type spatial patterns of maize inflorescence

Cell and Developmental Biology Lu Kang (Graduate Student)

Lu, Kang1 2
Han, Zhang1
Zichao, Li1 2
David, Jackson3
Fang, Yang1 2

1School of Agriculture and Biotechnology, Sun Yat-Sen University, Shenzhen, China, 518107
2National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China, 430070
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724

Postembryonic organogenesis in plants is orchestrated by spatiotemporal gene expression and regulatory networks in meristems, necessitating precise methods to characterize cellular fate transitions. Existing techniques–including single-gene in situ hybridization (ISH), single-molecule fluorescence in situ hybridization (smFISH), multiplexed error-robust FISH (MERFISH), and high-throughput methods like in situ sequencing (Stereo-seq, Visium HD) exhibit trade-offs in throughput, sensitivity, and tissue compatibility, lacking integrated robustness for multi-gene validation. Here, we present RCA-FISH, a robust, multiplexed in situ detection method optimized for formalin-fixed, paraffin-embedded (FFPE) plant tissues. RCA-FISH achieves sensitive detection of low-abundance transcripts across diverse tissues and enables simultaneous visualization of 30 genes in developing maize ear, revealing cell-type-specific co-expression patterns. Integrated with pci-Seq approach, it accurately maps cell types based on spatial expression signatures at single-cell resolution. This approach balances high sensitivity, multiplexing capacity, and broad tissue compatibility, addressing critical needs for scalable spatial gene expression analysis. Additionally, RCA-FISH bridges single-cell transcriptomics and spatial biology, providing a versatile tool to dissect plant organogenesis regulatory networks by overcoming bottlenecks in plant multi-gene in situ detection.

P138: Nodal root anatomical variation and allometric scaling in wheat

Cell and Developmental Biology Akshay Babasaheb Tawale (Graduate Student)

Tawale, Akshay B1
Schierenbeck, Matias1
Schneider, Hannah M1 2

1Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, Saxony anhalt, Germany 06466
2Georg-August University Göttingen, Lower saxony, Germany 37075

Nodal roots (adventitious roots) are critical to the structural support and nutrient uptake of cereal crops like wheat. In maize, anatomical differences have been observed across nodal roots depending on their emergence position (node number). We hypothesized that sequential nodal roots (older roots at basal stem positions and younger roots at more distal stem positions) in wheat would also exhibit distinct anatomical characteristics linked to their nodal position of emergence, and that these traits are under different genetic control. To test this, we initiated a Genome-Wide Association Study (GWAS) using a diversity panel of 190 hexaploid wheat genotypes evaluated in two field environments (spring and winter sowing) and controlled greenhouse conditions. Root samples were collected from four sequentially emerging nodes (node 1 to node 4, from the stem base towards the apex), with root sections taken 2 cm from the root-shoot junction for high-throughput anatomical imaging using the Rapid Anatomics Tool. A complementary hydroponics experiment with wheat roots confirmed that anatomical changes in roots from different nodal positions were not driven by tissue age at the time of sampling. Results revealed that anatomical variation in wheat roots across nodes is observed. Specifically, older roots exhibited a larger xylem vessel area compared to younger roots. Furthermore, allometric effects were evident, with anatomical traits scaling with the total root area, suggesting isometric growth within roots from a given node. Interestingly, younger roots showed a larger overall cross-sectional area than older roots, primarily driven by an increase in the root cortex area. These initial findings suggest distinct positional effects and allometric scaling in wheat nodal root anatomy, providing a foundation for ongoing GWAS to identify the genetic factors underlying root anatomical variations in different root classes.

P139: Northern teosinte - The artificial teosinte, which defies daylength limitations

Cell and Developmental Biology Tom Burges (Graduate Student)

Burges, Tom1
Salazar-Vidal, Miriam Nancy2
Davis, Danielle T3
Garberding, Andrea C3
Tracy, William F.3
Flint-Garcia, Sherry4

1Division of Biological Sciences; University of Missouri; Columbia, Missouri 65211, USA
2Division of Plant Science and Technology; University of Missouri; Columbia, Missouri 65211, USA
3Department of Plant and Agroecosystem Sciences; University of Wisconsin-Madison; Madison, Wisconsin 53706-1514, USA
4Plant Genetics Research Unit; U.S. Department of Agriculture-Agricultural Research Service; Columbia, Missouri 65211, USA

Northern Teosinte was created by W.C. Galinat, who crossed Guerrero teosinte x Gaspé Flint. The offspring of this cross was then backcrossed to teosinte multiple times. This artificial teosinte was already proven to flower and produce seeds under Massachusetts field conditions, however, no formal phenotyping had been performed. In this study, we performed a systematic characterization of Northern Teosinte in Missouri and a parallel study in Wisconsin. For the Missouri field evaluation, we planted 4 randomized blocks of Northern Teosinte together with Gaspé Flint, Zea mays ssp. parviglumis and Zea mays ssp. mexicana. In the greenhouse in Missouri, 5 plants were grown in the summer conditions; and 20 plants were grown in a growth chamber under long day conditions. One of the growth chamber plants was selected before flowering for selfing in isolation. The following traits were measured: tiller number, flowering time (first appearance of anthesis on the main stalk and first appearance on the tillers; first appearance of silking), germination rate, plant height, presence of naked kernels, seed shattering, number of nodes with ears, number of ears on the same node, number of ranks in ear, number of kernels per (row in) ear, ear length, and width. Overall, we determined that Northern Teosinte looks and behaves like teosinte in all aspects but flowering time. Northern Teosinte is able to flower and produce seeds under Missouri field and greenhouse conditions as well under long day conditions inside a growth chamber.

P140: Pcd1 controls kernel filling by promoting differentiation and cell death in the placenta-chalazal region

Cell and Developmental Biology Geng Sun (Graduate Student)

Sun, Geng1 2
Zhang, Jie1 2
Fontanet-Manzaneque, Juan B.1 2
Lee, Jimin1 2
Aesaert, Stijn1 2 3
Coussens, Griet1 2 3
Hoengenaert, Lennart1 2 3
De Rycke, Riet4
Doll, Nicolas M.5
Nowack, Moritz K.1 2

1Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent 9052, Belgium.
2VIB Center of Plant Systems Biology, VIB, Ghent 9052, Belgium.
3Crop Genome Engineering Facility, VIB, 9052 Ghent, Belgium.
4VIB Bioimaging Core, Ghent University, 9052 Ghent, Belgium.
5Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, CNRS, INRAE, F 69342, Lyon, France.

Kernel filling requires tight coordination between nutrient delivery by maternal tissues and nutrient uptake by filial tissues, particularly the endosperm. While the basal endosperm transfer layer (BETL) has been extensively characterized, the development and regulation of the adjacent maternal placenta–chalazal (PC) region remain poorly understood. Here, we identify Placenta-clalazal differentiation1 (Pcd1), a transcription factor preferentially expressed in the PC region, as a key regulator of PC differentiation. Loss of Pcd1 function results in a hyperplastic PC region that fails to acquire characteristic PC morphology, indicating a defect in differentiation. Notably, whereas PC cells in wild-type kernels undergo cell death during development, PC cells in pcd1 mutants remain viable, suggesting that Pcd1 regulates a developmentally programmed cell death process in the PC region. Consistent with a critical maternal function, pcd1 mutants exhibit a strong recessive phenotype: the majority of kernels on homozygous mutant cobs abort during early development, while a small fraction develop into severely dwarfed mature kernels. These findings indicate that proper PC differentiation and cell death are essential for normal kernel filling and viability. Together, our results identify Pcd1 as a central regulator of PC development and implicate programmed cell death in the optimization of nutrient transfer. This work advances our understanding of the maternal–filial interface and highlights the PC region as a key regulatory node in maize kernel filling.

P141: Rapid and selective peri-germ cell membrane breakdown during double fertilization

Cell and Developmental Biology Naoya Sugi (Postdoc)

Sugi, Naoya1
Millan-Blanquez, Marina2
Calhau, Andrea RM.2
Plagnard, Chloé2
Montes, Emilie2
Susaki, Daichi1 3
Ebine, Kazuo4
Kinoshita, Tetsu1
Thomas, Widiez2
Maruyama, Daisuke1

1Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan
2Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France.
3College of Science, Shizuoka University, Shizuoka, Japan
4Graduate School of Science and Engineering, Saitama University, Saitama, Japan

In flowering plants, immotile sperm cells are delivered to the female gametes through the pollen tube. During this transport process, the pair of sperm cells is enclosed by a vegetative cell–derived membrane, termed the peri-germ cell membrane. Only recently unified under a single name, this membrane remains functionally unexplored. However, the phospholipase NOT-LIKE-DAD (NLD), also known as MATRILINEAL (MTL) or ZmPLA1, which was identified as a key factor in haploid induction in maize, was shown to specifically localize to the peri-germ cell membrane, thereby drawing increasing attention to this atypical structure. . Immediately after pollen tube burst into the embryo sac, the peri–germ cell membrane must be rapidly and selectively removed to expose the sperm cell plasma membrane for successfull gamete fusion. However, the dynamics of peri-germ cell membrane breakdown and the trigger signals that initiate this process remain largely unknown. We established a maize line expressing NLD–mCitrine in the pollen vegetative cell and used it as a fluorescent marker for the peri-germ cell membrane to investigate the regulatory mechanism underlying its breakdown. When pollen grains were mechanically disrupted to release peri-germ cell membrane-enclosed sperm cells, we observed rapid peri-germ cell membrane breakdown outside the pollen grain. To identify the physiological conditions responsible for this phenomenon, we examined culture medium components and found that calcium acts as a trigger for peri-germ cell membrane breakdown. Recent live-cell imaging studies using calcium sensors have revealed Ca²⁺ spikes during double fertilization in Arabidopsis. Here, we discuss the molecular mechanism of Ca²⁺-induced peri-germ cell membrane breakdown and its potential role in the double fertilization.

P142: Single-cell transcriptomic profiling of a maize ploidy series with enhanced photosynthetic capacity

Cell and Developmental Biology Xiaowen Shi (Principal Investigator)

Yang, Yuxian1
Liu, Shuyi1
Xu, Yichun1
Birchler, James A.2
Shi, Xiaowen1

1College of Agriculture and Biotechnology, Zhejiang University, Hangzhou,China 310058
2Division of Biological Sciences, University of Missouri, Columbia, MO, USA 65211

Autopolyploids exhibit pronounced advantages over their diploid counterparts, including larger organs, cells, and biomass. While early physiological and phenotypic studies have documented the superior performance of autopolyploid maize, the molecular mechanisms driving this advantage remain poorly understood. In this study, we generated a maize ploidy series, comprising haploid, diploid, and tetraploid lines, derived from the B73 inbred background. Photosynthetic characterization demonstrated a positive correlation between ploidy level and key physiological metrics, including gas exchange rate, photosynthetic efficiency, and carbon assimilation capacity, indicating enhanced photosynthetic potential in autopolyploids. To investigate the molecular basis of this photosynthetic advantage, we generated a single-cell transcriptomic atlas of leaf tissues across the ploidy series. This dataset will provide a foundational resource for elucidating the gene regulatory networks and cellular mechanisms that underpin photosynthetic advantage in autopolyploid maize, offering new insights from a single-cell perspective.

P143: Spatial regulation of cell wall remodeling shapes maize digestibility under drought

Cell and Developmental Biology Ana López Malvar (Postdoc)

López-Malvar, Ana1
Collombel, Maia1
Lima, Stephen1
Guillaume, Sophie1
Mechin, Valerie2
Reymond, Matthieu1

1Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin for Plant Sciences (IJPB), 78000, Versailles, France.
2UMR AGAP Institute, University of Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France

Cell wall composition and structure are central to maize response under drought, and understanding how they relate to digestibility has important implications for breeding. Although increases in cell wall digestibility have been described under drought, it remains unclear whether this response depends on the specific localization of cell wall remodeling across tissues. We therefore asked whether increases in cell wall digestibility are driven by modifications in particular tissues.Using a combined histological and biochemical analysis, we found that drought reduced lignification in all tissue types, indicating changes in tissue organization . These anatomical changes were tightly correlated with biochemical alterations and strongly associated with increased in vitro cell wall digestibility. Our results show that variation in p-coumaric acid (pCA) content and its spatial distribution is a key driver of digestibility plasticity in response to drought. To characterize this response, we analyzed rind and pith tissues separately across eight genotypes: four plastic lines that increased digestibility by decreasing pCA under water stress, and four stable lines that showed little or no variation in either digestibility or pCA content. We found that cell remodeling occurs in the rind of both stable and plastic lines, but in the pith only in plastic genotypes. This demonstrates that increased whole-plant digestibility under drought is driven by cell wall modifications in the pith. In plastic genotypes, this response involves decreased lignification and p-coumaroylation in the pith accompanied by a compensatory increase in ferulic and diferulic acid cross-linking. By linking tissue-specific remodeling with digestibility plasticity, our findings provide markers that can support breeding efforts for improved forage quality and drought response.

P144: Spatiotemporal transcriptomics reveals ZmGRAS90 as an early determinant of maize aleurone and basal endosperm fate specification

Cell and Developmental Biology Hao Wu (Principal Investigator)

Cai, Xiaoyan1 2
Yang, Shuai1 2
Wu, Hao1 2

1State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization,Nanjing Agricultural University, Nanjing, China
2College of Agriculture, Nanjing Agricultural University, Nanjing, China

Maize endosperm not only provides the nutrients for embryo development and germination, but also is an important calory source for human diets and animal feeds. Early endosperm development sets up tissue architecture essential for later nutrient biosynthesis and storage. However, most studies have analyzed the endosperm development during the grain-filling stage, but regulatory mechanisms initiating tissue differentiation following cellularization remain poorly understood. In this study, we profiled spatial transcriptomes at 4, 6 and 9 DAP endosperms in single-cell resolution. This captured the transition from cellularization (4 DAP) to early differentiation (6 DAP) and finally to a defined tissue state before grain-filling (9 DAP). Our analysis revealed dynamic cell fate specification at the endosperm periphery: from a single, uniform progenitor cell cluster at 4–6 DAP resolved into two distinct aleurone (AL) and basal endosperm transfer layer (BETL) clusters by 9 DAP. This suggests that AL and BETL lineages may originate from a common progenitor population whose identity may be established as early as the cellularization stage. Especially, a transcription factor ZmGRAS90 is identified likely as an early regulator of this fate decision. ZmGRAS90 expression initiates specifically across the peripheral layer at 4 DAP, preceding the activation of canonical AL and BETL marker genes, AL9 and BETL9. By 6 DAP, its expression maximum shifts and co-localizes with BETL9 in the emerging BETL. At 9 DAP, ZmGRAS90 expression becomes tightly restricted to the boundary domain between the developing BETL and AL. This spatiotemporal progression suggests that ZmGRAS90 may play a dual role: first in broadly priming peripheral cell fate, and later in defining the spatial position and facilitating the differentiation of BETL. Understanding and potentially tuning early-stage regulators could allow us to optimize the fundamental architecture of maize kernels, with the ultimate goal of building better grains.

P145: Supervised clustering sheds light on the developmental trajectories of young inflorescence in barley

Cell and Developmental Biology Thirulogachandar Venkatasubbu (Postdoc)

Venkatasubbu, Thirulogachandar1
Walla, Agatha1
Lautwein, Tobias2
Lichtenberg, Stefanie3
Rutten, Twan4
Simon, Rüdiger5
von Korff Schmising, Maria1

1Heinrich Heine University, Faculty of Mathematics and Natural Sciences, Institute for Plant Genetics, CEPLAS, Düsseldorf, Germany
2Heinrich Heine University, Medical Faculty, Biological and Medical Research Center (BMFZ), Genomics and Transcriptomics Laboratory (GTL), Düsseldorf, Germany
3Heinrich Heine University, Medical Faculty and University Hospital Düsseldorf, Core Facility Flow Cytometry, Düsseldorf, Germany
4Leibniz Institute of Plant Genetics and Crop Plant Research, Structural Cell Biology, Gatersleben, Germany
5Heinrich Heine University, Faculty of Mathematics and Natural Sciences, Institute for Developmental Genetics, CEPLAS, Düsseldorf, Germany

Coordination among diverse shoot meristems is essential for optimizing cereal grain yield. In barley, a major cereal crop, the shoot apical meristem gives rise to leaves and tillers (vegetative axillary meristems) during the vegetative phase. Following the transition to the reproductive phase, it transforms into an inflorescence meristem that produces spikelets and florets (reproductive axillary meristems), which ultimately form grains. However, the developmental trajectories and identity transitions of these distinct shoot meristems remain poorly understood. Here, we employed multiplex single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) and single-nucleus RNA sequencing (snRNA-seq) on barley inflorescences at the early spikelet initiation stage. Using tissue-specific expression profiles obtained from smRNA-FISH, we reconstructed the developmental trajectories of the inflorescence meristem, leaf primordia, and axillary meristems. We further identified key regulatory genes associated with each lineage and functionally validated the roles of MANY NODED DWARF 1 and MANY NODED DWARF 6 in leaf and axillary meristem development. Collectively, our findings provide a high-resolution view of shoot meristem diversification in barley and represent a valuable resource for uncovering genetic determinants of cereal grain yield

P146: The EAL1 peptide controls embryonic meristem initiation in maize

Cell and Developmental Biology Guojing Jiang (Postdoc)

Jiang, Guojing1
Krohn, Nadia1
Wang, Lele1
Zhou, Liang-zi1
Dresselhaus, Thomas1

1Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, 93053 Regensburg, Germany

Maize (Zea mays L.) is a major cereal crop whose kernels are widely used for food and feed. A detailed understanding of its embryogenesis is essential for yield improvement and for its effective use in breeding programs. In contrast to the well-characterized embryogenic pattern of the model plant Arabidopsis thaliana, maize and other grasses show a higher degree of developmental complexity, including the formation of lineage-specific structures such as the coleoptile, scutellum, embryonic leaves, and a more elaborate embryonic shoot and root system. Here, we focus on the initiation and establishment of embryonic meristems in maize. We found that major transcriptome reprogramming occurs between the mid- and late transition stages, coinciding with the onset of embryonic organ differentiation. ZmEAL1 (Zea mays Egg Apparatus1-like1) encodes a peptide that is asymmetrically secreted to the cell wall of L1-layer cells at the adaxial region of the embryo proper at the early transition stage. This marks the site of stem cell niche initiation of the shoot apical meristem. Its gene is switched off after the mid-transition stage. We further found that it is a key regulator for meristem establishment: the Zmeal1 mutant fails to form a proper shoot–root axis. In summary, our findings provide new insights into the function of the grass-specific peptide ZmEAL1 in defining the outgrowth domain of meristematic cells and, thereby, in regulating the formation of the shoot–root system in embryos.

P147: The cis-regulatory evolution of GRASSY TILLERS1 (GT1)

Cell and Developmental Biology Hailong Yang (Postdoc)

Yang, Hailong1 2
Gallagher, Joseph P3
Lally, Kyra2
McKnight, Ronan2
Amundson, Kirk2
Brady, Eve2
De Neve, Amber2
Tang, Ben2
Facette, Michelle2
Bartlett, Madelaine1 2

1Sainsbury Laboratory, Cambridge University, Cambridge, England, United Kingdom CB2 1LR
2Department of Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA 01003
3Forage Seed and Cereal Research Unit, US Department of Agriculture, Agricultural Research Service, Corvallis, Oregon USA 97331

Plant form directly impacts crop yield. The evolution of form is thought to be driven largely by cis-regulatory evolution of conserved developmental genes, whereby gain, loss, or modification of cis-regulatory elements (CREs) alters gene expression. These cis-regulatory elements are often revealed as conserved noncoding sequences (CNSs). However, how CRE evolution modulates developmental gene function to impact trait variation remains poorly understood. To answer this question, we focus on GRASSY TILLERS1 (GT1), which encodes a Class I HD-ZIP transcription factor. GT1 is a pleiotropic domestication gene and represses growth in multiple developmental contexts, including tillers, floral organs, and axillary buds. A major domestication QTL upstream of maize GT1 (Prolificacy1.1/prol1.1) regulates variation in ear number, and includes multiple CNSs, suggesting that cis-regulatory variation at this locus might contribute to the evolution of branching. To test this, we made several CRISPR/Cas9 alleles in maize and brachypodium GT1 orthologs to examine how CNS evolution modulates GT1 gene expression to govern branching. By integrating evolutionary genomics, genome editing, and molecular assays, our research will provide fundamental insights into how CNS variation modulates developmental gene function in the evolution of plant form.

P148: The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency

Cell and Developmental Biology Zongliang Chen (Postdoc)

Chen, Zongliang1 6
Zhou, Jiaqi2 6
Galli, Mary1
Debernardi, Juan M.3 4
Dubcovsky, Jorge3 4
Jackson, David2
Gallavotti, Andrea1 5

1Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ, USA
2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA
3Department of Plant Sciences, University of California Davis, Davis, CA, USA
4Howard Hughes Medical Institute, Chevy Chase, MD, USA
5Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA
6These two authors contributed equally to this work

Transformation is a critical tool in plant genetics and functional genomics. Although recent advances have improved stable maize transformation, the molecular mechanisms underlying maize regeneration and its genotype dependency remain poorly understood. To address this, we developed a new transformation system based on morphogenic regulators to enhance efficiency in the maize B104 inbred line. This system combines the maize BABY BOOM transcription factor (ZmBBM/EREB53), driven by the tissue-specific pPLTP promoter, with the wheat GRF4–GIF1 chimera, driven by the maize ubiquitin promoter, alongside a modified QuickCorn protocol. This combination, termed “GGB”, enabled regeneration of transformed maize seedlings within approximately two months, achieving a transformation efficiency of ~20%. Importantly, overexpression of these morphogenic factors in B104 did not significantly affect development, eliminating the need to remove them after transformation. However, while female fertility remained intact, male fertility was partially compromised, resulting in skewed transmission through the male gametophyte. To evaluate the regeneration capacity of the GGB system in recalcitrant leaf tissues, we developed stable GRF-BBM (GB) and GGB transformants, both of which can regenerate whole plants from young leaves, demonstrating that BBM and GRF4 overexpression is sufficient to reprogram recalcitrant somatic cells for somatic embryogenesis and complete plant development. We further performed time-series RNA-seq analysis of somatic embryogenesis during leaf regeneration to identify novel genes involved in promoting somatic embryogenesis. Additionally, we generated fluorescent reporter lines to monitor auxin transport and response during the early stages of somatic embryogenesis. These genetic resources provide a powerful platform to dissect the molecular mechanisms of maize somatic embryogenesis and regeneration.

P149: The distributive germline: A developmental strategy to restrict the spread of new mutations

Cell and Developmental Biology Justin Scherer (Graduate Student)

Scherer, Justin1
Gaisie, Ahema1
Park, Sungjin1
Nelms, Brad1

1University of Georgia, Athens, Georgia, USA, 30602

We propose the Distributive Germline as a strategy to limit the spread of mutations among offspring. The Distributive Germline posits that plants derive their germline from multiple lineages established in the embryo, which are then radially organized in the meristem throughout development. We sequenced new mutations induced by transposable element insertions in pollen and offspring to provide evidence for the Distributive Germline. We find multiple deep lineages contributing to pollen and confirm that they are radially distributed, with some lineages detected before the first leaf diverged from pollen. Additionally, siblings share fewer insertions when generating crosses from the entire circumference of the tassel. We find parallels to the Distributive Germline across Eukaryotes, with other species also organizing germlines from multiple cell lineages early in development. @font-face {font-family:“Cambria Math”; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Aptos; panose-1:2 11 0 4 2 2 2 2 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:536871559 3 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:““; margin-top:0in; margin-right:0in; margin-bottom:8.0pt; margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:12.0pt; font-family:”Aptos”,sans-serif; mso-ascii-font-family:Aptos; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Aptos; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Aptos; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:“Times New Roman”; mso-bidi-theme-font:minor-bidi; mso-font-kerning:1.0pt; mso-ligatures:standardcontextual;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:“Aptos”,sans-serif; mso-ascii-font-family:Aptos; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Aptos; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Aptos; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:“Times New Roman”; mso-bidi-theme-font:minor-bidi;}.MsoPapDefault {mso-style-type:export-only; margin-bottom:8.0pt; line-height:115%;}div.WordSection1 {page:WordSection1;}

P150: The peri-germ cell membrane: A gateway to haploid embryo induction?

Cell and Developmental Biology Marina Millán Blánquez (Postdoc)

Millán Blánquez, Marina1
Calhau, Andrea1
Sugi, Naoya2
Jacquier, Nathanael1
Montes, Emilie1
Gilles, Laurine3
Widiez, Thomas1

1Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon1, CNRS, INRAE, Lyon, France
2Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan
3CIRAD, AGAP institute, Montpellier, France

In flowering plants, pollen grains exhibit a unique “cell(s) within a cell” organisation in which the two sperm cells are enclosed by a unique membrane, the peri-germ cell membrane (PGCM), and linked to the vegetative cell nucleus. This structure is rapidly dismantled upon pollen tube discharge for proper sperm cells release and fusion with female gametes, highlighting its essential role in double fertilisation.In our lab, we have identified the maize protein NOT-LIKE-DAD/MATRILINEAL/PHOSPHOLIPASE-A1 (NLD/MTL/ZmPLA1) as one of the few proteins localising exclusively to the PGCM. Interestingly, in maize, nld/mtl/pla1 mutants show severe pollen developmental defects and impaired double fertilisation resulting in the production of haploid embryos lacking the paternal genome. The haploid induction capacity of these mutant lines is routinely used by maize breeders, since haploid plantlets, after whole genome duplication, produce pure homozygous plants in just one generation, greatly improving breeding efficiency.To further elucidate PGCM function, we are investigating additional candidate proteins that may localise to this membrane and whose disruption could similarly affect pollen development and/or fertilisation. A promising candidate is a transmembrane H(+)-ATPase identified as an NLD interactor in immunoprecipitation assays. We have now generated genome-edited, Cas9-free maize lines for this protein and early segregation analyses reveal a strong transmission bias consistent with impaired pollen function, as only ~5% of homozygous mutants are recovered from selfed heterozygotes. Ongoing work is examining PGCM localisation of the ATPase:GFP fusion protein and whether disruption of this putative PGCM-associated protein can trigger haploid induction, and whether combined mutations (e.g., nld/mtl/pla1) may have synergistic effects.We believe that addressing these questions has the potential to provide a deeper insight into the fundamental bases of sexual reproduction in plants and holds promise for advancing plant breeding strategies, such as in planta haploid induction.

P151: The role of ZmARF28 in leaf initiation and development in maize

Cell and Developmental Biology Bianca Tasha Ferreira (Graduate Student)

Ferreira, Bianca T1
Richardson, Annis E1

1Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

All grasses have a distinct conserved leaf shape, orientation and angle. The developmental mechanisms that control leaf traits are, in part, controlled by auxin pathways. Auxin is a phytohormone that is a key regulator of development underpinning cell fate, organ initiation, and architecture. Auxin plays a role in controlling leaf development through a family of functionally distinct DNA-binding Auxin Response Factors (ARFs). One such example in maize is ZmARF28. ZmARF28 is a class-B ARF (classified as a repressor) that, when mutated, leads to changes in leaf number, phyllotaxy and midrib formation. The mutation results in a Ser-to-Asn amino acid substitution in a loop region in the DBD resulting in an over-accumulation of the ZmARF28 protein. A transcriptomic dataset comparing Trf seedlings treated with auxin versus control, revealed both up and down regulated genes within 30minutes of treatment. This suggests that ZmARF28 may have a more complex role, potentially acting as both an activator and repressor of gene expression. By mining published tissue-specific and single-cell RNA-seq datasets coupled with dual-luciferase reporter assays we have started to elucidate the gene regulatory network that ZmARF28 acts in during maize leaf initiation and development. Identifying key genes interacting with ZmARF28 to regulate the precise balance between auxin, spatial gene expression, and leaf development in maize will further our understanding of how auxin pathways and class-B ARFs function in maize.

P152: Transcriptomic control of heterosis manifestation during lateral root initiation in maize

Cell and Developmental Biology Annika Meyer (Graduate Student)

Meyer, Annika1
Hochholdinger, Frank1
Yu, Peng2

1Institute of Crop Science and Resource Conservation, Crop Functional Genomics, University of Bonn, 53113 Bonn, Germany
2TUM School of Life Sciences, Plant Genetics, Technical University of Munich, 85354 Freising, Germany

Heterosis is a concept describing the superior performance of hybrid offspring relative to their inbred parents. Multiple prominent traits that are influenced by heterosis are visible aboveground, such as biomass accumulation and growth rate, while belowground, heterosis also markedly shapes root system architecture. In young maize seedlings lateral root density displays significant heterosis. The spatial positioning of lateral roots along the parental root axis determines the three-dimensional structure of the root system and influences the efficiency of nutrient and water uptake. In maize, phloem-pole pericycle cells provide a stem cell niche that gives rise to lateral roots. To understand how heterosis shapes lateral root formation, we profiled the transcriptomes of phloem-pole pericycle cells from recombinant inbred lines with contrasting lateral root densities and from their backcrosses to the founder lines Mo17 and B73. We harvested phloem-pole pericycle cells via laser capture microdissection. The exploration of gene expression patterns in this specific cell type that are associated with heterosis is a major focus of our study. The understanding of these patterns will potentially aid in shaping effective crop root systems for future agricultural challenges.

P153: Transformation and genome editing of the maize model Line Gaspé Flint 1.1.1

Cell and Developmental Biology Silvio Salvi (Principal Investigator)

Gualtieri, Ruggero1
Camerlengo, Francesco1
Collier, Ray2
Lor, Vai S2
Walter, Nathalie2
Forestan, Cristian1
Guo, Shengjie1
Tuberosa, Roberto1
Kaeppler, Shawn M2
Salvi, Silvio1

1Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy, I-40127
2Wisconsin Crop Innovation Center, University of Wisconsin, Middleton, WI, USA, WI 53562

Efficient in vitro transformation and plant regeneration systems are essential for applying genome editing to functional genomics studies; however, these processes remain strongly genotype-dependent in maize. The early-flowering inbred line Gaspé Flint 1.1.1 (GF111), derived from a Canadian northern flint landrace, is characterized by high homozygosity, a rapid generation cycle, ease of hand pollination, good fertility, and minimal requirements in terms of space and resources. The aim of this study was to establish an efficient Agrobacterium–mediated transformation and in vitro regeneration protocol for GF111. To enhance tissue culture responsiveness, GF111 explants were transformed using a vector overexpressing Wuschel-like homeobox 2a (Wox2a), a gene known to promote embryogenic culture formation. Using this transformation and editing platform, we targeted Vgt1, a major cis-regulatory element influencing the downstream flowering-time gene ZmRap2.7. Specifically, we aim to generate a series of targeted deletions to dissect the functional subdomains of the Vgt1 locus. The development of an efficient transformation and genome editing system for GF111 will substantially increase the utility of this line as a model for functional genomics studies of maize development and environmental adaptation.

P154: Two semi-dominant mutants acting in a common pathway regulate maize leaf angle

Cell and Developmental Biology Guan Zhang (Graduate Student)

Zhang, Guan1 2
Wu, Qingyu1 2

1Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences; Beijing, China, 100081
2National Nanfan Research Institute, Chinese Academy of Agricultural Sciences; Sanya, China, 572024

Maize leaf angle is a key agronomic trait that determines plant architecture and adaptation to high-density planting. However, dense planting can exacerbate biotic stress and often requires a fine balance between optimal architecture and immune regulation. Previous studies have shown that the semi-dominant mutant Liguleless narrow (Lgn-R) displays a reduced leaf angle and compact plant architecture, a phenotype likely associated with autoimmunity. LGN encodes a plasma membrane–localized receptor kinase, and its kinase activity is essential for its biological function. However, the downstream client(s) of LGN remain unknown, limiting mechanistic dissection of its roles in leaf development and immune regulation and constraining its application in crop improvement. Here, we cloned a novel semi-dominant mutant, designated Lgn-like (LgnL), which exhibits phenotypes similar to Lgn-R, including reduced leaf angle and autoimmunity. Notably, during a TurboID-mediated proximity labeling screen for LGN-interacting proteins, we detected a strong proximal association between LGN and LGNL. This interaction was further validated by split-luciferase complementation assays and bimolecular fluorescence complementation (BiFC). The identification of LGNL reveals a previously unrecognized component of the LGN signaling pathway and provides new insights into the molecular mechanisms by which LGN coordinates leaf development and immune responses. Moreover, this work offers valuable genetic resources and potential targets for breeding maize varieties with improved tolerance to dense planting and enhanced disease resistance.

P155: feminized upright narrow1 is a novel nuclear disordered protein that mediates brassinosteroid and oxylipin biosynthesis

Cell and Developmental Biology Jazmin Abraham-Juarez (Postdoc)

Abraham-Juarez, Jazmin1 2
Vajk, Angus1
Yuan, Peiguo3
Bertolini, Edoardo4
Kolomiets, Michael3
Eveland, Andrea4
Hake, Sarah1
Chuck, George1

1University of California Berkeley, Albany, CA USA 94710
2Sainsbury Laboratory University of Cambridge, 47 Bateman Street Cambridge UK CB2 1LR
3Department of Plant Pathology and Microbiology Texas A&M University; College Station, TX, USA 77843
4Donald Danforth Plant Science Center, St. Louis, MO, 63132

Although most flowers are bisexual, some have evolved to be monecious, having staminate and pistillate flowers on different parts of the plant. In the male staminate flowers of maize this is achieved through the biosynthesis of two hormones, oxylipins such as jasmonic acid that are known to suppress growth, and brassinosteroids that promote it. How the proper balance between growth inhibition and promotion is coordinated by these two hormones in male flowers is unknown. Here, we analyze a novel sex-determination mutant, feminized upright narrow1 (fun1), that lacks pistil abortion in male flowers and exhibits highly reduced leaf auricles. We cloned fun1 by chromosome walking and found that it encodes a highly disordered nuclear protein that is specifically expressed in the L1 layers of several tissues and organs, including the degenerating carpels of male flowers. Mass spectrometry, yeast two-hybrid, and bimolecular fluorescence complementation analysis show that FUN1 physically interacts with lipid biosynthetic proteins such as ZMLOX5, which produces oxylipins, and NANA2, which produces brassinosteroids. This shows that FUN1 represents a new epidermal-specific integrator that balances the biosynthesis of opposing hormones to bring about unique developmental outcomes in different regions of the plant. We present a two-step model where FUN1 first promotes brassinosteroid synthesis to form the epidermis, later allowing it to become competent to respond to an oxylipin-mediated pistil abortion signal.

Computational and Large-Scale Biology

P156: AI-ready genomics and multi-omic functional annotation at MaizeGDB

Computational and Large-Scale Biology Carson Andorf (Principal Investigator)

Haley, Olivia C.1
Tibbs-Cortes, Laura E.1
Harding, Stephen F.1
Cannon, Ethalinda K.1
Portwood II, John L.1
Gardiner, Jack M.2
Woodhouse, Margaret R.1
Andorf, Carson M.1 3

1USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, 819 Wallace Rd., Ames, Iowa, United States 50011
2Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA
3Department of Computer Science, Iowa State University, 2434 Osborn Dr, Ames, Iowa, United States 50014

The integration of Artificial Intelligence (AI) into computational biology is improving agricultural research by enabling the extraction of biological insights from increasingly complex datasets. As a primary resource for the maize (Zea mays L.) community, the Maize Genetics and Genomics Database (MaizeGDB) is proactively establishing an AI-ready infrastructure designed to support machine learning applications. This strategic initiative involves standardizing multi-omic datasets, generating precomputed embeddings from state-of-the-art DNA and protein language models, and providing reproducible workflows via GitHub. New AI-driven functionalities include zero-shot variant-effect scoring and genome browser tracks for nucleotide conservation, which facilitate the interpretation of functional significance across the genome.These AI resources are deeply integrated with an expanded suite of functional annotation tools and visualization tracks within the MaizeGDB genome browser. New Syntenome tracks provide whole-genome alignments with other grasses and lifted gene models to support comparative analyses and gene model validation. Users can also access unmethylated regions, aligned protein fragments from the Maize Peptide Atlas, and high-confidence protein structure predictions from AlphaFold and ESMFold. To capture structural and functional diversity across lineages, the database incorporates pangenome and pan-gene annotations from the Nested Association Mapping (NAM) founder genomes. Furthermore, over 600 epigenetic and DNA-binding datasets provide critical regulatory context, including open chromatin regions and histone modifications. The integration of these data with tools such as SNPVersity and PanEffect allows researchers to navigate from sequence variation to functional impact. By combining AI-ready data with a robust framework for inter-species and cross-species comparisons, MaizeGDB empowers the maize genetics community to accelerate gene function discovery and the development of improved maize varieties.

P157: Using the MaizeGDB genome browser to evaluate gene model annotation quality and gain functional insights

Computational and Large-Scale Biology John Portwood (IT-Specialist)

Portwood II, John L.1
Tibbs-Cortes, Laura1
Haley, Olivia C.1
Woodhouse, Margaret1
Cannon, Ethalinda1
Gardiner, Jack2
Andorf, Carson1

1USDA-ARS Corn Insects and Crop Genetics Research Unit, Ames, IA 50011, USA
2Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA

MaizeGDB genome browsers provide an integrated suite of tools, datasets, and resources that enable users to evaluate gene model annotation quality and gain functional insights into maize genomics. MaizeGDB now includes Syntenome tracks that support whole-genome alignments with other grasses and lifted gene models, enabling comparative analyses that aid gene model validation and highlight conserved and diverged regions. These tracks incorporate unmethylated regions, aligned protein fragments from the Maize Peptide Atlas, and confidence metrics from protein structure prediction. Together with more than 300 gene expression datasets, these resources support robust comparative analyses within maize and across species. Additional evidence includes protein structure alignments from AlphaFold and ESMFold and full-proteome alignments for more than 20 species via UniProt and Phytozome, providing further validation and functional context. Alternative annotations from NCBI, Helixer, and Mikado offer complementary or confirmatory support, while transcription start site predictions from CAGE data refine transcriptional start site annotations. Functional interpretation is further enhanced through curated annotations, including UniProt descriptions, Gene Ontology terms, AI-based predictions, and transcription factor annotations from Grassius. Pangenome and pan-gene annotations from the Nested Association Mapping (NAM) population founder genomes and other high-quality maize assemblies enable exploration of structural and functional variation across maize lineages. More than 600 epigenetic and DNA-binding datasets, including transcription factor binding sites, open chromatin regions, and histone modifications, add regulatory context. The genome browser also integrates tools for visualizing DNA variation, along with forward genetics resources such as UniformMu, BonnMu, and Ac/Ds insertions. Tight integration with gene model pages, BLAST, SNPVersity (variant visualization), and PanEffect (variant effect visualization) supports seamless navigation and analysis. Collectively, these resources help users critically assess gene models, interpret functional evidence, and accelerate maize genetics and genomics research.

P158: Benchmarking software tools for identifying common plant metabolites in LC-MS datasets

Computational and Large-Scale Biology Elliot Braun (Graduate Student)

Braun, Elliot1 2
Thompson, Addie2 3
VanBuren, Robert2 3 4
Grotewold, Erich4 5

1Genetics and Genome Sciences Program, Michigan State University
2Plant Resilience Institute, Michigan State University, Michigan State University
3Department of Plant, Soil & Microbial Sciences, Michigan State University
4Department of Plant Biology, Michigan State University
5Department of Biochemistry and Molecular Biology, Michigan State University

Unraveling the plant metabolome remains an ongoing challenge, and untargeted approaches that aim to detect a wide range of metabolites can be valuable in unpacking some of the many questions that remain. However, the structural diversity of plant metabolites complicates downstream structure prediction and annotation, and understanding how current software performs on known metabolites is a critical knowledge gap. In this study, a group of 37 reference standards composed mostly of phenolic compounds have been subjected to untargeted liquid chromatography with tandem mass spectrometry (LC-MS/MS). Four top-of-the-line software packages have then been evaluated in their performance for detecting the standards and subsequently annotating them. The findings prove somewhat unexpected, with correct annotations ranging from only 7/37 to 36/37 depending on the software and parameters used. Notably, higher positive annotation rates typically correspond to a higher number of false positive annotations for a given mass feature. The findings from this software benchmarking have been applied to a biological dataset of sorghum grain extracts from 333 accessions in the Sorghum Association Panel, highlighting a valuable use case for optimized metabolomics processing workflows. Previously unknown mass features were given high-confidence annotations, and unknown metabolites were given tentative annotations. This work provides updated information on the state of metabolomics processing workflows and recommendations for future researchers aiming to find trustworthy metabolic signals in untargeted datasets.

P159: Beyond chromatin: DNA replication timing becomes a novel determinant of meiotic recombination landscapes

Computational and Large-Scale Biology Quinn Johnson (Graduate Student)

Johnson, Quinn1
Markham, Emily2
Pawlowski, Wojciech P.1

1Cornell University, Ithaca, NY, 14853
2NC State University, Raleigh, NC, 27695

The uneven distribution of meiotic crossovers (COs) along chromosomes reflects regulatory mechanisms that shape recombination landscapes. COs, in maize, are strongly suppressed in pericentromeric regions and the processes underlying this bias remain unclear. Since meiotic recombination is initiated by double-strand breaks (DSBs) and follows DNA replication during S-phase, we hypothesized that replication timing (RT), together with chromatin features, contributes to shaping recombination landscapes. We found that genomic regions replicating early in S-phase favored CO formation, but DSB formation occurred less than expected, where most DSBs occurred in mid-replicating regions that infrequently harbor COs. Within early-replicating DNA, replication efficiency, the probability that an origin fires concomitantly across cells, was higher at CO sites than at DSB sites, indicating that stable origin firing characterizes chromosomal domains permissive for CO formation. COs require a chromatin environment of low nucleosome occupancy and DNA hypomethylation, whereas DSB formation is not dependent on undermethylated DNA. Notably, RT patterns were not correlated with DNA methylation, indicating that RT influences recombination events independently of chromatin state. In comparison, Arabidopsis exhibits similar RT distributions to maize but lower DNA methylation and no pericentromeric CO suppression. Strikingly, both DSBs and COs in Arabidopsis were enriched in early-replicating regions of high replication efficiency, a pattern distinct from maize. As RT cannot be manipulated experimentally in plants, we used simulations to test whether changing RT patterns alters recombination landscape. These simulations suggested that RT had stronger effects on DSB placement, likely because DSB formation is less constrained by DNA methylation. Whereas CO placement, which is restricted by DNA methylation and CO interference, appeared less strongly affected by RT. Our findings highlight RT as a previously underappreciated but profoundly important determinant of recombination landscape. Although temporally separated, DNA replication and meiotic recombination patterns overlap, suggesting an intriguing mechanistic coordination.

P160: Decoding the agronomic genome: Foundation models for precision trait discovery

Computational and Large-Scale Biology Javier Mendoza Revilla (Research Scientist)

Boshar, Sam1
Evans, Benjamin1
Tang, Ziqi1
Picard, Armand1
Adel, Yanis1
Lorbeer, Franziska K.2 3
Rajesh, Chandana1
Karch, Tristan1
Sidbon, Shawn1
Emms, David1
Mendoza-Revilla, Javier1
Al-Ani, Fatimah1
Seitz, Evan1
Schiff, Yair1 4
Bornachot, Yohan1
Hernandez, Ariana1
Lopez, Marie1
Laterre, Alexandre1
Beguir, Karim1
Koo, Peter5
Kuleshov, Volodymyr4
Stark, Alexander2 3
de Almeida, Bernardo P.1
Pierrot, Thomas1

1InstaDeep, London, UK
2Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria
3Medical University of Vienna, Vienna BioCenter (VBC), Vienna, Austria
4Cornell Tech, New York, USA
5Cold Spring Harbor Laboratory, New York, USA

The rapid advancement of DNA foundation models is transforming our ability to decode complex genomic patterns, shifting the field beyond simple sequence analysis to the prediction of regulatory syntax and cellular phenotypes.Here, we present the Nucleotide Transformers (NTv3), a suite of models designed to bridge the gap between sequence diversity and functional outcomes. While existing models often separate sequence learning from functional data, NTv3 integrates ~10 trillion nucleotides from 100,000 species with dense functional annotations across 18 animals and 6 major plant lineages. This unified architecture has established a new state of the art in structural annotation and variant effect prediction, facilitating application for precision agriculture. We will present benchmarking results showing that NTv3 outperforms domain-specific models across key agronomic tasks. For example, in predicting molecular phenotypes in maize, NTv3 achieves state-of-the-art accuracy. For transcriptional regulation, across 23 diverse tissues, NTv3 attains a mean coefficient of determination (R²) of 0.737 for gene expression prediction, substantially exceeding the AgroNT baseline of 0.535. For proteomic abundance, when modeling protein levels across eight tissues, NTv3 demonstrates strong generalization performance and achieves a mean R² of 0.460, nearly doubling the predictive performance of the prior state of the art, which reports an R² of 0.233. These results illustrate how unified sequence-function architectures can provide a framework for high-resolution in silico mutagenesis and the systematic discovery of traits.By accurately decoding non-coding regulatory syntax, NTv3 provides a robust framework for identifying and optimizing gene editing targets for complex agronomic traits. These open-source models will accelerate translational research, offering new solutions for precision agriculture and synthetic biology.

P161: Evolutionary dynamics and co-adaptation of root traits and microbiome function in Poaceae drought tolerance

Computational and Large-Scale Biology Xiaolin Lyu (Graduate Student)

Lyu, Xiaolin1 2 3
Yu, Peng1 2

1Emmy Noether Group Root Functional Biology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany
2Plant Genetics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
3Crop Functional Genomics, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany

Drought stress, a major threat to global crop productivity, severely limits plant survival, especially in arid environments. Drought tolerance is a key adaptive trait conserved across Poaceae grasses, which remains poorly understood, particularly in relation to its interactions with the rhizosphere microbiota. While recent studies have focused on the genetic mechanisms driving desiccation tolerance in Poaceae, the role of rhizosphere microbes in this process is still underexplored. In this study, we investigated drought tolerance across 227 Poaceae species, representing a wide range of phylogenetic lineages and geographic distributions. We employed an integrated approach combining host phylogeny, biogeographic traits, and rhizosphere microbiome profiling to uncover the evolutionary basis of desiccation tolerance. Using 16S rRNA sequencing, we systematically characterized the rhizosphere microbiome to identify drought-resilient microbial taxa and explore their functional associations with plant desiccation tolerance. We also isolated bacteria from the rhizosphere of 30 species of grasses. Our findings revealed phylogenetically conserved patterns of microbial recruitment linked to drought adaptation. By correlating microbial community composition with plant drought resilience, this study provides a new insight into the microbiome-mediated drought tolerance hypothesis.

P162: Exploring the maize transcriptional regulatory landscape through large-scale profiling of transcription factor binding sites

Computational and Large-Scale Biology Zeyang Ma (Principal Investigator)

Huo, Qiang1
Zhang, Ziru1
Ma, Zeyang1
Song, Rentao1

1State Key Laboratory of Maize Bio-breeding, College of Agronomy and Biotechnology, China Agricultural University, Beijing, China, 100193

Understanding gene regulatory networks (GRNs) is essential for improving maize yield and quality through molecular breeding. However, the lack of comprehensive transcription factor (TF)-DNA interaction data has hindered accurate GRN predictions, leaving regulatory mechanisms poorly understood. To address this gap, we performed large-scale profiling of maize TF binding sites, generating a robust dataset for 513 TFs and identifying 394,136 binding sites. By integrating these data with chromatin accessibility and gene expression profiles, we constructed a highly accurate maize GRN. This network comprises 397,699 regulatory interactions, which we further partitioned into distinct modules across six major tissues. Additionally, we employed machine-learning algorithms to enhance the prediction accuracy of gene functions and key regulators. This study provides the largest experimental collection of TF binding sites in maize to date and serves as a valuable resource for dissecting gene function and advancing crop improvement.

P163: Genes driving maize stem responses to water deficit: from expression to phenotype

Computational and Large-Scale Biology Sylvie Coursol (Research Scientist)

Baudry, Kevin1 2 3
Chaignon, Sandrine4
Brasero Pardow, Nerea2 3 4
Jacquemot, Marie-Pierre4
Paysant-Le Roux, Christine2 3
Beaubiat, Sébastien4
Pateyron, Stéphanie2 3
Tardieu, François5
Welcker, Claude5
Turc, Olivier5
Vitte, Clémentine1
Joets, Johann1
Cabrera-Bosquet, Llorenç5
Méchin, Valérie1
Martin, Marie-Laure2 3 6
Coursol, Sylvie4

1Université Paris-Saclay, INRAE, CNRS, AgroParisTech, Génétique Quantitative et Evolution - Le Moulon, Gif-Sur-Yvette, 91190, France
2Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif-sur-Yvette, France
3Université de Paris Cité, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif-sur-Yvette, France
4Université Paris-Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant Sciences, 78000, Versailles, France
5Université de Montpellier, INRAE, LEPSE, Montpellier, France
6Université Paris-Saclay, INRAE, AgroParisTech, UMR MIA Paris-Saclay, 91120, Palaiseau, France

Water deficit strongly affects the performance of forage maize by reducing biomass production and altering digestibility1. Recent studies have shown that this stress can induce substantial shifts in cell wall composition, including phenolic compounds such as p-coumaric acid2,3. These effects are highly genotype-dependent, reflecting contrasted biochemical adjustments that influence both water stress sensitivity and forage digestibility. However, the molecular mechanisms linking water deficit, cell wall remodeling, and digestibility remain poorly understood. In the context of climate change and increasing water scarcity, identifying genetic determinants of forage digestibility under water-limited conditions is essential for developing resilient cropping systems. To address this challenge, seven contrasting maize genotypes were characterized using indoor high-throughput plant phenotyping (HTP) platforms under well-watered and moderate water deficit conditions. HTP traits were combined with measurements of digestibility, cell wall composition, and transcriptomic profiles collected from three stem internodes per plant. Linear mixed models were applied to identify genes whose expression was associated with HTP, digestibility, and cell wall composition traits, focusing on their contribution to genotype × water condition interaction variance. The results identify promising candidate genes for forage quality traits and provide a valuable genomic resource to support the improvement of digestible yield in forage maize under limited water conditions.1Main, O. et al. (2023) Precise control of water stress in the field reveals different response thresholds for forage yield and digestibility of maize hybrids. Front Plant Sci. doi: 10.3389/fpls.2023. 2López-Malvar, A et al. (2025) Genotype-dependent response to water deficit: increases in maize cell wall digestibility occurs through reducing both p-coumaric acid and lignification of the rind. Front Plant Sci. doi: 10.3389/fpls.2025.1571407. 3Main, O. et al. (2025) Targeting enhanced digestibility: Prioritizing low pith lignification to complement low p-coumaric acid content as environmental stress intensity increases. PLoS One. doi: 10.1371/journal.pone.0338077.

P164: Genome assembly and flowering-time regulation in the model maize line Gaspé Flint 1.1.1

Computational and Large-Scale Biology Silvio Salvi (Principal Investigator)

Guo, Shengjie1
Gualtieri, Ruggero1
Camerlengo, Francesco1
Vitte, Clementine2
Joets, Johann2
Piazzi, Guglielmo1
Tuberosa, Roberto1
Salvi, Silvio1

1Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy, I-40127
2Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE – Le Moulon, Gif-sur-Yvette, France, F- 91190

We assembled a high-quality genome of Gaspé Flint 1.1.1 (GF111), a super-early, small-stature maize model inbred line (Salvi et al. 2022, Journal of Plant Registrations 16:152–161), using the PacBio long-read sequencing platform. BUSCO evaluation indicated high completeness of the coding space (96.6%), with 88.7% single-copy and 7.9% duplicated genes. To support gene annotation and downstream transcriptomic analyses, Illumina RNA-seq was performed on 22 distinct GF111 tissues, generating approximately 490 Gb of transcriptomic data. Based on transcriptomic evidence and orthologous protein information, a custom ab initio gene prediction model was trained. Transcriptomic, proteomic, and ab initio evidence were then integrated using the MAKER pipeline to produce a comprehensive, high-confidence gene annotation. We further conducted detailed analyses of gene structure and expression for several flowering-time–related genes, including ZmCCT, ZmRap2.7 and Vgt1, ZCN8, and others, identifying genomic and transcriptional features that contribute to the early flowering phenotype of GF111. Transposable element annotation confirmed the presence of a MITE insertion at the Vgt1 locus, consistent with previous reports linking this insertion to early flowering. Additionally, differential expression analyses based on comparisons with a diverse panel of maize inbred lines revealed expression differences in flowering-time genes, providing further insights into the genetic mechanisms underlying flowering-time variation. [Shengjie Guo is supported by EUROPEAN MSCA-COFUND “FutureData4EU” Doctoral Programme Grant Agreement n. 101126733]

P165: HAIant: Symbiotic integration between humans and artificial intelligence

Computational and Large-Scale Biology Yunlong Zhang (Graduate Student)

Zhang, Yunlong1 2
Wang, Yuming3
Zheng, Zhilong1
Wu, Hao3
Zeng, Xiangfeng1
Huang, Qishuai1
Wei, Wendi3
Fan, Youkun1
Zhou, Tao1
Zhu, Guanzhen1
Han, Rui1
Zheng, Ruozhuo3
Zhang, Hongwei4
Zhang, Chuang5
Wang, Yi1
Liu, Xiangguo5
Li, Weifu3
Feng, Zaiwen3
Li, Lin1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, Wuhan 430070, China
3College of Informatics, Huazhong Agricultural University, Wuhan 430070, China
4Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
5Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Institute, Jilin Academy of Agricultural Sciences, Changchun 130124, China

Abstract: [Objective] Artificial intelligence (AI) and its associated applications bear human-level capabilities in numerous aspects and are used extensively across many and diverse domains. Nevertheless, concerns regarding the security of people’s data and thoughts while using AI-related applications remain. [Method] HAIant was invented for the alleviation of AI concerns. It is built using PyQt5 for the development of its graphical user interface (GUI), with Ollama used to manage the deployment and execution of locally hosted large language models (LLMs). [Result] Here, we devise a platform called ‘HAIant’ (Human + AI + ant, Humans use the power of artificial intelligence and exploit the advantages of swarm intelligence like ants). HAIant allows people to integrate the computational power of AI with personal data and thinking logic for the development of personal AI, and to work together using swarm-intelligence principles inspired by ant colonies. This framework leverages privately deployed large language models (LLMs) to incorporate user-specific data, work records, and agent-specific plugins to extend the capabilities of the LLM. As a proof of concept, we develop ‘biological HAIant’, which can help record biological experiments, carry out bioinformatics analysis, and even the communication and sharing of biological data with the help of AI. Biological HAIant can eventually assemble a manuscript based on the wet and dry biological experiments by users. [Conclusion] HAIant is local, personal, and most importantly, safe for the augmentation of personal intelligence. Beyond AI agents, which are dedicated to completing a task or event for humans, the era of a human-oriented HAIant is coming to nurture both personal and swarm intelligence. As AI continues to be improved, HAIant is expected to grow and possibly co-exist with AI, largely assured by security in the manner of local human-orentied AI models.Key words: HAIant, AI, Security, LLM

P167: Integrating proteomics data into a genome-scale metabolic model to predict metabolic fluxes in maize leaf

Computational and Large-Scale Biology Maëla Sémery (Graduate Student)

Sémery, Maëla1
PETRIZZELLI, Marianyela2
PRIGENT, Sylvain3 4
BLEIN-NICOLAS, Mélisande1
DILLMANN, Christine1

1GQE-Le Moulon, IDEEV, 12 route 128, 91190 Gif-sur-Yvette
2Sanofi, 82 avenue Raspail, 94250 Gentilly, France
3UMR 1332 BFP, 71 avenue Édouard Bourlaux, 33882 Villenave d'Ornon, France
4MetaboHUB, PHENOME-EMPHASIS, 71 avenue Edouard Bourlaux, 33882 Villenave d’Ornon, France

Maize is one of the most important cereal crops in the world, but it is highly sensitive to drought, with yield losses of up to 50% depending on the developmental stage. Thus, developing drought-tolerant varieties, especially for agroecological transition, is crucial and requires a deeper understanding of adaptation mechanisms. Advances in high-throughput phenotyping now enable the characterization of many individuals at the transcriptome, proteome, and metabolome levels across different environments. However, linking genetic or molecular changes to phenotypic adaptations remains challenging. Data integration methods, particularly those using constraint-based metabolic models, can predict metabolic fluxes and provide new insights into the most active metabolic pathways under varying environmental conditions. Several approaches predict fluxes across metabolic networks by applying constraints to solve stationary state equations. Some use omics data to estimate fluxes that best fit experimental observations. Among these, Petrizzelli et al. proposed a method using proteomic data to constrain flux predictions, which has been tested on a simplified model of central carbon metabolism in yeast. To investigate maize adaptation to drought, we adapted Petrizelli et al.’s approach to a curated metabolic model of the maize leaf. First, we performed a structural analysis of this model to show that the network reactions are clustered into meaningful metabolic pathways. We then connected the metabolic model to proteomic data obtained from the leaves of 254 maize hybrids grown under two contrasted irrigation conditions to predict metabolic fluxes. The resulting flux values discriminate between the two irrigation conditions, indicating that our flux predictions are biologically meaningful. The interpretation of flux variation in response to drought is ongoing. This method can easily be used to analyze the impact of other abiotic stresses on the primary metabolism of maize, thereby increasing our understanding of the mechanisms by which maize respond to these stresses.

P168: Machine learning for removal of technical bias signals in high-throughput transcriptomics datasets

Computational and Large-Scale Biology Teague McCracken (Graduate Student)

McCracken, Teague1 2
Gordon, Max1 2
Scroggs, Taylor3
Nelms, Brad3
Williams, Cranos1 2

1Department of Electrical and Computer Engineering, North Carolina State University, Raleigh, NC, USA 27606
2N.C. Plant Sciences Initiative, North Carolina State University, Raleigh, NC, USA 27606
3Department of Plant Biology, University of Georgia, Athens, GA, USA 30601

Plate-based high-throughput transcriptomics (HTT) enables large-scale transcription factor (TF) perturbation experiments for inferring genetic regulatory networks (GRNs). GRNs can capture TFs’ roles in regulating key biological processes like regeneration and meiosis. In plants like Zea mays, identifying the regulatory interactions that control transcription is critical for developing new applications of cellular reprogramming, such as enhanced regeneration or in vitro plant nurseries. Perturbing elements like TFs or drugs enable the identification of causal signals, constraining the solution space of regulatory networks that explain the observed data. Consequently, increasing the number of perturbations can improve confidence in inferred regulatory relationships. However, the large scale of plate-based HTT experiments leads to a mixture of causal signals, correlations, noise, and technical bias that arises from cells responding to variability in the experimental environment. Conditions can differ throughout the data collection process due to sample pooling, batching, and edge effects. Therefore, an approach is needed to separate bias from perturbation response signals in HTT data to increase accuracy in downstream network inference. In this study, we evaluate a regularized linear model and a nonlinear general additive model for bias removal and preservation of perturbation response signals on our maize TF perturbation data and analogous human drug perturbation HTT data. The two models perform comparably in reducing associations between sources of technical bias and data structure at global and local scales. The linear model overcorrects the data, amplifying global linear patterns and causing greater shifts in local data structure. In contrast, the nonlinear model optimally balances the removal of bias and preservation of perturbation response signals, improving perturbation separability and drug dose-consistent differential expression patterns. Our study demonstrates that by modeling features as separable additive functions, the nonlinear model provides greater flexibility in precisely estimating technical bias effects, enabling unbiased GRN inference and enhancing data quality.

P169: Maize genomic prediction: Integrating genomics and transcriptomics for trait analysis

Computational and Large-Scale Biology Ally Schumacher (Graduate Student)

Schumacher, Ally M.1
Sitar, Sidney2
Gomez Cano, Lina3
Brottlund, Donielle2
Newton, Linsey2
Turkus, Jon4 5 6
Schnable, James C.4 5 6
Grotewold, Erich3
Thompson, Addie2

1Department of Plant Biology; Michigan State University, East Lansing, MI 48824, USA
2Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI, 48824, USA
3Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, MI, 48824, USA
4Quantitative Life Sciences Initiative, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA
5Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA
6Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA

Tar spot disease in maize, caused by the fungal pathogen Phyllachora maydis, can reduce yield by up to 40% depending on infection severity. Polyphenolic compounds have been preliminarily linked to tar spot resistance, suggesting a relationship between compound accumulation and plant disease response. Pigmentation traits, such as anther and silk coloration, are influenced by polyphenolic compounds and may be connected to shared genetic pathways and regulatory networks underlying both aesthetic and disease resistance traits. Genome-wide association studies (GWAS) are being conducted to investigate these traits, focusing on identifying shared pathways and regulatory mechanisms that link phenolic compound accumulation, pigmentation, and tar spot resistance. Significant single nucleotide polymorphisms (SNPs) identified through GWAS will be incorporated into genomic prediction models to evaluate their utility in predicting phenotypic variation in pigmentation traits and phenolic accumulation. To complement this, transcriptome wide association studies (TWAS) and whole genome comparative network analyses (WGCNA) are being conducted to identify genetic loci and expression networks associated with tar spot severity. These analyses leverage a Wisconsin Diversity Panel evaluated over multiple years and locations to study regulatory networks influencing phenolic compound accumulation in relation to plant susceptibility to tar spot disease. By combining GWAS-identified loci with co-expression patterns from WGCNA and trait-associated transcripts from TWAS, this study aims to identify candidate genes associated with both pigmentation and phenolic traits. This integrative approach is expected to enhance the discovery of functional variants and improve genomic prediction accuracy, particularly for breeding applications targeting tar spot disease resistance.

P170: Missing heritability of 6 ecophysiological traits of maize in response to water deficit captured by integration of phenomic, proteomic, and genomic data

Computational and Large-Scale Biology Marie-Laure Martin (Principal Investigator)

Djabali, Yacine1 2 4
Rincent, Renaud4
Blein-Nicolas, Mélisande4
Martin, Marie-Laure1 2 3

1Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif sur Yvette, France
2Université Paris Cité, Institute of Plant Sciences Paris-Saclay (IPS2), 91190, Gif sur Yvette, France
3Université Paris-Saclay, AgroParisTech, INRAE, UMR MIA Paris-Saclay, 91120, Palaiseau, France
4Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE-Le Moulon, 91190, Gif-Sur-Yvette, France

The increase in the amount of genomic data is dramatically improving our understanding of genetic determinism of phenotypic traits through genome-wide association studies (GWAS). However, the genetic determinism of complex traits is generally not fully unraveled,mainly due to a lack of statistical power. The development of omics technologies now provides a means of identifying QTLs with indirect effects on complex phenotypic traits by establishing relationships between these traits, intermediate molecular traits, and genetic polymorphisms. We implemented an innovative systems biology approach integrating the genetic determinism of molecular traits through the combination of genome-wide association studies and network inference. At each step of the integration process, a multi-environment mixed model was used to estimate the contribution ofgenomic regions contribute to the biological variance, defined as the sum of the genotype variance and the genotype x water deficit interaction (GxW) variance. We applied our approach on a multi-omics dataset acquired from 254 maize hybrids grown under well-watered and water-deficit conditions, to link six ecophysiological traits with proteomic and genomic data. Our results show that the QTLs underlying variations in protein abundance capture a part of the biological variance of the six ecophysiological traits. We observed a synergy between loci identified in the two watering conditions and loci associated with plasticity indices calculated from the two conditions. Finally, we identified a protein with an unknown functionfor five of the six ecophysiological traits.

P171: Model, predict, generate: The utility of machine learning models in angiosperm genomics

Computational and Large-Scale Biology Ana Berthel (Research Scientist)

Berthel, Ana1
Zhai, Jingjing1
Liu, Zong-Yan2
Miller, Zachary R.3
McMorrow, Sarah1 4
Monier, Brandon1
Gokaslan, Aaron5
Czech, Eric6
Cannon, Betsy6
Marroquin, Edgar5
Hsu, Sheng-Kai1
Chen, Szu-Ping2
Stitzer, Michelle1
Romay, M. Cinta1 2
Pennell, Matt7
Kuleshov, Volodymyr5
Buckler, Edward S.1 2 3

1Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA 14853
2Section of Plant Breeding and Genetics, Cornell University, Ithaca, NY USA 14853
3USDA-ARS, Ithaca, NY, USA 14853
4Boyce Thompson Institute, Ithaca, NY, USA 14853
5Department of Computer Science, Cornell University, Ithaca, NY, USA 14853
6Open Athena AI Foundation, New York, NY, USA 10001
7Department of Computational Biology, Cornell University, Ithaca, NY, USA 14853

Machine learning models have demonstrated the capacity to process complex patterns and relationships in many fields, including biology. Here we present three applications of machine learning to plant genomics: PlantCAD2, GeneCAD, and MLImpute.PlantCAD2 is a DNA foundational language model trained to capture patterns of conservation in flowering plants. It improves upon the original PlantCAD model with a longer context window of 8,192 base pairs, a more efficient Mamba2 architecture, and an expanded training set of 65 diverse angiosperm genomes. PlantCAD2 surpasses the Evo2 model in 10 of 12 zero-shot tasks using 10 times fewer parameters. Its zero-shot scores can be used without additional training to predict variants that have a functional impact within the genome, or the model can be fine-tuned for specific tasks such as predicting chromatin accessibility and gene expression. Fine-tuning a foundational model requires less task-specific data than training from scratch and often produces models with better cross-species transferability. Therefore, models like PlantCAD2 can transfer knowledge from well-studied model organisms like Arabidopsis to other plant species. PlantCAD2 is also useful as a component of larger models such as the genome annotation tool GeneCAD. GeneCAD predicts complete gene models using only DNA sequence, allowing for accurate annotation of assemblies without the need to collect extensive transcriptomic or proteomic data. GeneCAD leverages PlantCAD2’s diverse pre-training to achieve robust results on unseen clades and performs better at reproducing the reference annotations of five held-out benchmark species than both BRAKER3 and Helixer. Finally, MLImpute is a pangenome-based machine learning tool for imputing genotypes from low-coverage sequencing data. It generates a set of variants based on homology to high-quality assemblies in the pangenome by modeling the process of genetic recombination, and can be used to impute haploid or diploid genotypes. Keywords: Machine Learning, Artificial Intelligence, DNA Language Model, Genome Annotation

P172: Phylogenetic study of Helitron polymorphism in Fungi, Plantae, or Animalia Kingdoms

Computational and Large-Scale Biology Bethany Olive (Graduate Student)

Olive, Bethany1
Du, Charles1

1Montclair State University; Montclair, NJ, USA

Transposable elements, or “jumping elements,” are powerful agents of evolution in eukaryotic DNA. Helitrons are an emerging family of Class 2 transposons discovered through computational analysis. These polymorphic elements are capable of gene capture and have shown recent movement in many genomes, despite being found as non-autonomous, fragmented sequences. Captured genes of interest are Heat Shock Elements (HSEs) involved in the activation of an organism’s heat shock response, as effects of climate change are prevalent. The transposition and activation mechanism for this movement remains speculative and understudied. This study investigates Helitron movement through comprehensive phylogenetic analysis within three kingdoms. Utilizing comparative genomic techniques, this analysis aims to reveal patterns in genetic movement in response to environmental change. Understanding this movement can predict how plants will react to global climate change, and the impact this may have on our agricultural future. This knowledge can also help identify and develop genetic mutations that are heat-resistant to protect crops and produce.

P173: Plastochron regulation in maize: Spatial transcriptomics reveals transcriptional changes in the plastochron1 mutant.

Computational and Large-Scale Biology Lotte Van de Vreken (Graduate Student)

Van de Vreken, Lotte1 2
Werbrouck, Stan1 2
Demuynck, Kirin1 2
Nelissen, Hilde1 2

1Ghent University, Department of Plant Biotechnology and Bioinformatics, Technologiepark 927, 9052 Ghent, Belgium
2VIB Center for Plant Systems Biology, Technologiepark 927, 9052 Ghent, Belgium

The maize plastochron1 (pla1) mutant exhibits a shortened plastochron (accelerated leaf initiation) and reduced plant growth. This remarkable phenotype suggests a role for PLA1 in regulating leaf initiation and growth in the maize shoot apical meristem (SAM). The SAM is a highly complex structure where stem cells gradually differentiate into plant organs in a spatially coordinated manner. However, the transcriptional mechanisms by which PLA1 influences these developmental processes remain unclear. To investigate how PLA1 affects plant architecture at the molecular level, we applied spatial transcriptomics to visualize the expression of 500 genes in wild-type and pla1 mutant shoot apices. Our analysis showed that PLA1 is expressed at the boundary between undifferentiated and differentiated cells, suggesting a role in regulating cell division and organ initiation. Developmental trajectory analysis tracing transcriptional changes from stem cells to differentiated cells revealed overlaps of PLA1 with stem cell markers such as Knotted1 and differentiation markers such as YABBY14, further supporting its regulatory function. Comparisons between wild-type and pla1 mutants revealed significant transcriptional changes, particularly in auxin metabolism, indicating an important role for PLA1 in (hormonal) regulation of the SAM. These findings visualize the spatial and temporal changes in the transcriptional regulation of pla1 mutant SAMs, showcasing spatial transcriptomics and its downstream data-analysis as a valuable tool for studying plant development.

P174: Predicted protein 3D structures provide essential insights into the genetic architecture underlying phenotypic diversity in maize

Computational and Large-Scale Biology Shuai Wang (Graduate Student)

Wang, Shuai1 2
Khaipho-Burch, Merritt3
Johnson, Lynn C.4
Miller, Zachary R.4
Bradbury, Peter J.5
Speed, Doug6
Allen, William J.7
Romay, M. Cinta4
Xue, Jiquan1
Buckler, Edward S.3 4 5
Ramstein, Guillaume P.6
Song, Baoxing1 2

1Key Laboratory of Maize Biology and Genetic Breeding in Arid Areas of the Northwest Region, College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China
2Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong 261325, China
3Section of Plant Breeding and Genetics, Cornell University, Ithaca, New York 14853, USA
4Institute for Genomic Diversity, Cornell University, Ithaca, New York 14853, USA
5Agricultural Research Service, United States Department of Agriculture, Ithaca, New York 14853, USA
6Center for Quantitative Genetics and Genomics, Aarhus University, Aarhus 8000, Denmark
7Texas Advanced Computing Center, University of Texas at Austin, Austin, Texas 78758, USA

Variation in protein 3D structures reflects genetic variation and contributes to phenotypic diversity, yet its underlying genetic mechanisms remain unclear. To investigate the relationship between protein 3D structure and phenotype, we predict the 3D structures of 795,649 proteins from 26 maize (Zea mays L.) inbred lines using AlphaFold2. Population genetics analysis of these protein 3D structures reveal that buried residues held greater genomic evolutionary rate profiling (GERP) scores than exposed residues, indicating that buried residues are under stronger purifying selection. The design of the maize nested association mapping population makes it possible to utilize haplotype information and protein 3D structural variation to reveal the molecular mechanisms linking genetic diversity and phenotypic variation for a population with about 5000 individuals. Associating protein 3D structure variation with phenotypes (structure-based proteome-wide association study [PWAS]) identifies 14.2% more (96 vs. 84) significant proteins compared with associating protein sequence with phenotypes (sequence-based PWAS) using 32 agronomic traits. Moreover, structure-based PWAS identifies 24 additional significant proteins unique to predicted structures, whereas sequence-based PWAS identifies 12 additional significant proteins. Structure-based proteome-wide predictions (PWPs) improve genomic prediction accuracy by an average of 3.8% compared with sequence-based PWPs. In general, predicted protein 3D structures represent a powerful approach for understanding the natural diversity of protein haplotypes.

P175: Predicting complex phenotypes using multi-omics data in maize

Computational and Large-Scale Biology Madison Creach (Graduate Student)

Creach, Madison1 3 4
Schnable, James2
Thompson, Addie3 4
VanBuren, Robert1 3 4

1Department of Plant Biology, Michigan State University, East Lansing, MI, USA
2Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, USA
3Plant Resilience Institute, Michigan Sate University, East Lansing, MI, USA
4Department of Plant, Soil & Microbial Sciences, Michigan State University, East Lansing, MI, USA

Understanding and predicting complex traits in plants remains a fundamental challenge due to the emergent nature of most phenotypes and their dependence on genetic, regulatory, and environmental interactions. Accurate prediction of traits and identification of underlying genetic elements has broad applications for plant breeding, systems biology, and biotechnology. We tested if multi-omic datasets could improve predictive accuracy of 129 diverse maize phenotypes across nine environments using genomic markers, field based transcriptomic data from two locations, and drone-derived phenomic data of vegetative indices. We trained and compared linear (rrBLUP) and nonlinear (support vector regression) models using single- and multi-omics inputs. Multi-omics models consistently outperformed single-omics models for most traits, with genomic and transcriptomic inputs contributing distinct biological features. Phenomic features alone yielded the lowest predictive power but improved predictions for specific trait categories like root architecture. Transcriptomic datasets enabled cross-environment prediction, demonstrating that gene expression patterns from one field site could accurately predict traits measured in another. Environment-specific expression of benchmark flowering time genes highlighted the value of transcriptomics in capturing genotype-by-environment (G×E) interactions not detectable through genomic data alone. These findings demonstrate that integrating transcriptomic and phenomic data with genotypes enhances trait prediction, improves model generalizability across environments, and provides deeper insight into the genetic and regulatory architecture of agriculturally important traits in maize.

P176: Regulatory response of maize to water deficit mediated by cis-regulatory elements

Computational and Large-Scale Biology Thomas-Sylvestre Michau (Graduate Student)

Michau, Thomas-Sylvestre1
Mary-Huard, Tristan1
Fagny, Maud1

1Unversité Paris-Saclay, INARe, CNRS, AggroParis Tech; GQE - Le Moulon; Gif-sur-Yvette, France, 91160

Climate change is intensifying summer droughts in Europe, significantly affecting maize growth and yield. In response to water deficit, maize regulates gene expression through signaling pathways. Transcription factors (TFs) interacts with cis-regulatory elements (CREs), including proximal (promoters) and distal (dCREs, enhancers and silencers) ones. The latter are responsible for the spatio-temporal regulation of gene expression, and can integrate environmental cues. However, identifying their target genes remains challenging, as dCREs can act over long genomic distances and often exhibit condition-specific regulatory activity, complicating their functional characterization. Regulatory network inference approaches provide a powerful framework to address this issue by integrating both proximal and distal CREs across multiple conditions to untangle regulatory relationship and their specificity.A panel of five maize inbred lines was selected, including the reference line B73, chosen for their genetic, phenotypic and stress-response diversity. Transcriptomic data were obtained for five tissues: ear, internode, leaf, silk and tassel, in 2 watering conditions, water deficit and control condition. For each line we inferred tissue- and condition-specific networks using the NETZOO suite.For each genotype, we observed that only a subset of tissues exhibited significant regulatory changes, and that the identify of the tissues involved in water deficit response vary between lines, coherently with phenotypic observations. We identified tissue-specific differentially regulated genes showing significant gene ontology enrichment for tissue-specific functions like photosynthesis in leaves and internodes or stamen formation in tassel, and diverse response to stress : oxidative stress, water deprivation, abscisic acid. Regulatory alterations were related to structural network modifications highlighting the role of TFs from ERF family and associated CRE in the response to water deficit. Overall, our findings reveals both common and line-specific CRE-mediated regulatory responses to drought across maize lines, underlying the interest of regulatory network inferences for characterizing these regulatory regions.

P177: Shortcuts to success: how large-scale genotyping benefits the maize community

Computational and Large-Scale Biology Corrinne Grover (Research Scientist)

Grover, Corrinne E1
Hufford, Matthew B1
Ross-Ibarra, Jeffrey2
Woodhouse, Margaret R3
Andorf, Carson M3 4

1Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa, USA, 50011
2Department of Evolution and Ecology, Genome Center, and Center for Population Biology, University of California, Davis, CA 95616, USA
3USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA 50011, USA
4Department of Computer Science, Iowa State University, Ames, IA 50011, USA

Maize is an incredibly versatile species, whose accessions are adapted to a variety of conditions and whose genomes consequently harbor variation useful in modern crop improvement. The maize community research mirrors this versatility, with projects spanning the full breadth of available germplasm. Recently, the Maize Genetics and Genomics Database (MaizeGDB) released SNPversity 2.0, an easy-to-use platform cataloging genomic variation from thousands of diverse maize lines, thereby facilitating variant identification and discovery between specific lines and B73. With SNPversity, users can easily pinpoint variants in their favorite line, compare those to B73 (and other genotyped lines), and explore the putative effects of that variation. Here we present the latest update to SNPversity (v2.1), which includes genotypes for over 2700 accessions and integrates functional predictions from PlantCAD. We provide practical examples demonstrating how SNPversity can accelerate maize research for the community, and provide an overview of the genotyping pipeline that will be annually run to jointly genotype many thousands of maize lines. Finally, we invite the community to suggest useful accessions or features for the 2026 SNPversity update (v2.2).

P179: Towards next-generation bioinoculants: Machine learning-assisted characterization of PGPB for maize

Computational and Large-Scale Biology Sulamita Santos Correa (Research Scientist)

Santos Correa, Sulamita1
Gitahy, Patricia1
Schultz, Júnia2
Soares Vidal, Márcia1
Zilli, Jerri Edson1
Soares Rosado, Alexandre3
Simões de Araújo, Jean Luis1

1Embrapa Agrobiologia (Brazilian Agricultural Research Corporation), Seropedica, Rio de Janeiro, Brazil.
2Nuclear Energy Center for Agriculture, University of São Paulo, Brazil.
3King Abdullah University of Science and Technology (KAUST), Jeddah, Saudi Arabia.

One of the main challenges for cropping has been the supply of Nitrogen (N), which, in addition to being a high-cost input, has low use efficiency by plants. Experiments with Plant Growth-Promoting Bacteria (PGPB) associate with maize plants have shown increased N efficiency. Despite the expansion of inoculant for maize in the market, the number of bacterial species in commercial inoculants is limited. Thus, it is necessary alternatives to expand the characterization of new bacterial species with the potential to promote maize growth, thereby increasing the availability of strains for the formulation of bioinoculants. Therefore, it is important the identification of highly competitive bacteria, capable of utilizing a wide variety of mechanisms to promote plant development and with capacity to nitrogen fixation, even under stress conditions such as high temperature and drought. The huge genomic sequences allow the selection of strains with PGP traits by identifying genomic regions related to plant-microorganism interactions. Additionally, the use of artificial intelligence (AI) enables the analysis of large datasets to identify bioactive molecules, genes, and specific regulatory regions of PGPB. This approach is essential for applying synthetic biology tools, such as genetic editing. Regarding this, in this study we applied three supervised and pre-trained machine learning tools, DeepGoPlus, Random Forest and PGPB finder, to characterize bacteria isolates from Brazilian and Antarctica soils as potential PGPB of maize. Bacteria such as Azospirillum baldaniorum and Herbaspirillum seropedicae were applied for validation with known functions. The pipeline allowed the integration of protein structures, combined domains, evolutionary signatures, genes and genomic contexts and identified several strains with potential to promote maize growth. Ongoing studies include in vitro evaluation of theses strains and inoculation in maize plants under greenhouse conditions. The use of these approaches will enable the development of second-generation multifunctional inoculants with a greater capacity to promote plant growth in tropical conditions.

Cytogenetics

P180: Cytogenetic analyses of mutants of class I and class II crossover regulators in maize

Cytogenetics Seijiro Ono (Postdoc)

Ono, Seijiro1
Ono, Misato1
van der Heide, Max1
Schnittger, Arp1

1Department of Developmental Biology, Institute for Plant Science and Microbiology, University of Hamburg, Ohnhorststr. 18, Hamburg, Germany, 22609

Formation of crossovers (COs) is a fundamental meiotic process that enables the exchange of genetic material between homologous chromosomes. COs are essential not only for generating genetic diversity through the recombination of parental alleles, but also for ensuring fertility by physically linking homologous chromosomes to promote balanced segregation at metaphase I. Despite the agricultural importance of maize, the mechanisms by which COs are designated and regulated in this species remain poorly understood, and it is unclear to what extent models derived from other organisms apply to maize. We recently optimized maize genetic transformation methods, enabling systematic functional analysis of meiotic genes. To elucidate the formation, distribution, and regulation of COs in maize, we first targeted components of the class I CO pathway, defined by the ZMM proteins and responsible for interference-sensitive COs that dominate in many plant species. Surprisingly, all zmm mutants analyzed (hei10, zip4, mer3 and msh4) exhibited only semi‑fertile phenotypes and retained more than 50% of wild-type CO levels. Given previous estimates suggesting that approximately 80% of COs in wild-type maize are interference-sensitive and therefore expected to be class I, these findings suggest that maize possesses a unique compensatory mechanism that maintains CO number despite disruption of the ZMM pathway. To further investigate this phenomenon, we generated additional mutants affecting other potential CO regulators, including class II CO endonucleases and anti‑CO factors, and combined them with zmm mutations. Preliminary results from these higher‑order mutant analyses will be presented, and potential regulatory mechanisms underlying the distinctive CO fate determination system in maize will be discussed.

P181: Engineering a maize mini-B chromosome as a platform for site-specific gene integration

Cytogenetics Jasnoor Singh (Graduate Student)

Singh, Jasnoor1
Swyers, Nathan1
Graham, Nathaniel1
Cody, Jon1
Gao, Zhi1
Albert, Patrice1
Liu, Hua2
Kelly, Jacob1
Yang, Bing2
Birchler, James1

1Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA
2Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA

In maize, random transgenic integration can make gene stacking difficult, especially when combining several features into a single line. An alternate platform for targeted gene insertion and repeated trait stacking without interfering with endogenous loci is provided by engineered mini-chromosomes produced from maize B chromosomes. A telomere-mediated truncating construct, pNT1, was transformed into maize carrying B chromosomes and a truncated miniB was recovered with a terminal transgene carrying a landing pad for phiC31 Integrase site specific recombinase together with a GFP marker, which allows the minichromosome to be followed phenotypically. The pNT1 construct was designed together with donor amendment plasmids to conduct gene stacking. The recovered miniB was crossed to Cre recombinase to remove the Bar selection marker, which positioned the miniB for reuse of Bar selection during subsequent addition to the chromosome. In parallel, an Integrase construct was recovered for which the Bar selection marker could be excised with FLP recombinase and simultaneously activate a GFP gene. Exposure of the Integrase transformant to FLP removed Bar and activated GFP, which also allows the transgene to be followed phenotypically. The function of the Integrase was confirmed by callus bombardment of an inverted DsRed construct flanked by attP and attB sites that upon recombination will activate the gene, which was found to be the case. These results point to an improved mini-B chromosome as a platform that facilitates targeted cargo acceptance via phiC31-mediated recombination. This work lays the groundwork using synthetic chromosome platforms in maize for introduction of genes onto an independent chromosome with the ability for gene stacking.

P182: Genome-Wide investigation of recombination in the presence of maize B chromosomes

Cytogenetics Malika Sharma (Graduate Student)

Sharma, Malika1
Yang, Hua1
Liu, Jian2
Boadu, Frimpong2
Cheng, Jianlin2
Albert, Patrice1
Birchler, James1

1Division of Biological Sciences, University of Missouri, Columbia, MO 65211
2Department of Electrical Engineering and Computer Science, University of Missouri, Columbia, MO 65211

B chromosomes (Bs) are supernumerary, dispensable chromosomes found in many species, including maize. Although not required for development, Bs in maize have been shown cytologically to increase meiotic recombination, particularly in heterochromatic regions. It has been proposed that the B chromosome evolved this effect to ensure its own segregation in meiosis given its smaller size and being heavily heterochromatic with the effect spilling over to the A chromosomes. To investigate this effect on the genome level we constructed high-resolution crossover (CO) maps from six reciprocal backcross maize populations generated from (B73/W22) × B73 crosses carrying 0, 2–3, or 6 Bs. Low-coverage sequencing and haplotype inference revealed dosage and sex-specific effects. In male meiosis, increasing B number elevated genome-wide CO frequency and promoted multi-CO bivalents, with rare high-CO class (6 COs) detected only in 6B males. In contrast, female meiosis did not show a significant change in recombination with increasing B dosage. These findings demonstrate that Bs reshape the recombination landscape in maize. Interestingly, the male specific effect might have evolved for proper meiotic segregation of the B immediately preceding the other aspects of its drive mechanism consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B containing sperm.

P183: Inheritance and segregation of heterochromatic knobs in an F2 progeny from highly endogamic inbred lines

Cytogenetics Ana Laura Marassi Maronezi (Graduate Student)

Marassi Maronezi, Ana Laura1

1Luiz de Queiroz College of Agriculture - University of São Paulo (ESALQ/USP)

Heterochromatic knobs are cytologically visible blocks of heterochromatin, consisting of tandem repeats of 180-bp and 350-bp sequence families, accounting for up to 8% of the maize genome. The presence and variability of heterochromatin knobs across all known maize inbred lines, traditional varieties, and hybrids suggest a strong selective pressure for their maintenance. Inbred lines such as C103 and Mo17 are known for their low knob content. As part of an effort to develop a knob-free (knoblessness) inbred line for future studies, we mapped knob frequencies in an F2 progeny from a C103 x Mo17 cross. While Mendelian segregation (1:16) was expected for a knobless individual, several mechanisms can cause preferential segregation of these structures. In 40 analyzed plants, 15 showed the most frequent configuration: 0+ for K8L and ++ for K9S. Configurations with intermediate quantities consisted of 8 individuals with 00 on K8L and 0+ on K9S, 9 with 0+ on K8L and 0+ on K9S, and 6 with 00 on K8L and ++ on K9S. In a smaller frequency, 2 individuals had ++ on K8L and ++ on K9S. In this sample, no plants were observed with 00 for both K8L and K9S, nor with ++ on K8L and 00 on K9S. These preliminary results suggest distorted segregation for the knob on the short arm of chromosome 9. Therefore, although the analysis will be expanded to a larger number of plants, distortion in K9S segregation is observed in the sample analysed. This transmission distortion indicates possible non-Mendelian and preferential segregation. These findings reinforce forces influencing heterochromatin inheritance, highlighting the need to investigate their impact on the maize genome. Understanding these patterns may provide insights into fundamental genetics and the origin and evolution of maize karyotypes.

P184: Maize knob heterochromatin and chromosomal features: A cytoinformatics framework

Cytogenetics Tiago Corrêa (Graduate Student)

Corrêa, Tiago E.1
Mondin, Mateus1

1CYNGELA, Department of Genetics, Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, SP, Brazil

Heterochromatic knobs are prominent cytological landmarks in maize (Zea mays ssp. mays) chromosomes and are primarily composed of satellite repeats K180 and TR-1. Although knobs are routinely described by classical cytogenetics, comparisons across multiple high-quality genome assemblies can help standardize knob localization and other chromosomal landmarks in a consistent, karyotype-oriented framework. Despite long-standing discussion of knobs in relation to genome size and chromosome architecture, linking knob-associated repeat landscapes to karyotype features remains challenging to assess systematically within modern inbred panels. Here, we report initial results from an ongoing study using a cytogenetics-focused bioinformatics approach called cytoinformatics to generate digital karyotypes from telomere-to-telomere assemblies. For each genome, we infer cytogenetic descriptors (chromosome length, arm ratio, and centromere position) and map key markers via sequence-similarity searches, including Cent-C–enriched centromeric arrays, knob-associated satellites (K180 and TR-1), and ribosomal DNA loci (45S and 5S rDNA). Marker coordinates are clustered into representative chromosomal regions and integrated into visualization scripts to produce idiograms directly comparable to standard cytogenetic maps. We apply this workflow to assemblies from 26 NAM founder inbreds plus Mo17, using B73 and Mo17 as references for cross-line comparisons. Across this closely related panel, total genome sizes differ by at most 3.49% (Mo17: 2.179 Gb; Il14H: 2.103 Gb), and most chromosomes exhibit similar sizes across lines, consistent with their relatedness. This observed range likely underestimates genome-size variation across broader maize diversity. Preliminary comparisons indicate that digital idiograms reproduce the expected marker locations and overall chromosome morphology, while also revealing line-specific deviations in morphometric parameters (e.g., arm ratio) and marker distributions that can guide targeted follow-up studies. Overall, cytoinformatics provides a practical bridge between classical cytogenetics and genome assemblies for scalable surveys of knob heterochromatin and chromosomal organization.

P185: Mapping the second trans-acting factor(s) required for the B chromosome nondisjunction

Cytogenetics Hua Yang (Research Scientist)

Yang, Hua1
Gilbert, Nathan1
Liu, Jian2
Cheng, Jianlin2
Birchler, James A.1

1Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA 65211
2Department of Electrical Engineering and Computer Science, University of Missouri, Columbia, MO 65211

In maize, the B chromosome enhances its own transmission through frequent nondisjunction (NDJ) during the second pollen mitosis, producing sperm with two or zero B chromosomes. This drive mechanism requires multiple B-encoded trans-acting factors. One factor, gene 666, encodes a degenerate F-box–associated protein but whose Kelch-repeat domain is predicted to remain functional. Structural prediction and mutational analyses show that the N-terminal portion of the Kelch region is dispensable for NDJ, whereas a single amino acid substitution that likely disrupts the Kelch structure abolishes NDJ. These results suggest that the Kelch domain mediates a critical protein-interaction function required for B-chromosome NDJ. Because 666 alone cannot account for the process, we re-investigated classic TB-10L translocations to identify additional factors previously postulated. Internal B-chromosome deletions generated by combining different B-10L and 10L-B chromosomes revealed that the triB-10L7/TB-10L9 combination reduces NDJ to 33%, compared with 73% in wild type. This deletion likely removes a proximal euchromatic region harboring a second essential factor. NDJ estimates in earlier studies were confounded by the presence of tertiary trisomic kernels. To improve accuracy, we are using a PCR-based method to exclude tertiary trisomics from euploid kernels with true B-chromosome deletions. These results will clarify whether other NDJ-required trans-acting factors reside within the proximal region between 10L7 and 10L9. In parallel, RNA-seq data from various tissues containing B chromosomes from Liu et al., 2025 and Hloušková et al., 2025 are being analyzed to assess gene expression patterns in this region. Together, these approaches will help to identify the second (and possibly additional) factor(s) and advance our understanding of the molecular mechanism underlying B-chromosome NDJ.

P186: Molecular analysis of Barbara McClintock’s tiny fragment chromosome

Cytogenetics Taylor Isles (Graduate Student)

In 1978 McClintock described a fragment chromosome that could rearrange itself as well as become inserted into or attached to other chromosomes. McClintock attributed this to an “X component” on the fragment chromosome. She later produced a 1.6 Mbp version called “tiny fragment”. This chromosome contained a centromere, near which she mapped the X component, attached to a short section of chromosome arm 9S. The marker genes on tiny fragment, Shrunken1 and Bronze1, showed evidence of loss, presumably by the failure of tiny fragment to be included in a developmental lineage. However, kernels were also observed that indicated a loss of function of either Sh1 or Bz1 alone. McClintock noted that many cases of Bz1 mosaicism were typical of a silenced state with activation in small sectors in the endosperm and that these patterns could occur in clusters on ears. We confirm the observations of McClintock regarding kernel expression of Sh1 and Bz1. We determined the structure and gene content of Tiny Fragment through DNA sequencing and observed elevated gene expression of genes across Tiny Fragment via RNAseq. In previous propagation of the chromosome a variant was isolated that was routinely unstable more so than the original. Unstable Tiny Fragment plants exhibit a mosaic phenotype for bz1 in both the plant and endosperm tissue. RNAseq analysis of gene expression of Unstable Tiny Fragment plants reveals markedly lower levels for the resident genes than in the original Tiny Fragment lines but still higher than in control plants without any Tiny Fragment suggesting that it is lost during development at a higher frequency. We are investigating whether the structure or other features of this derivative are responsible for the different behavior.

P187: Neotelomere formation in maize

Cytogenetics Kempton Bryan (Graduate Student)

Bryan, Kempton1
Dawe, Kelly R1 2

1Department of Genetics; University of Georgia, Athens, GA, 30602
2Department of Plant Biology; University of Georgia, Athens, GA, 30602

Prior work has shown that it is possible to increase chromosome number in maize by chromosome fission. When recombination occurs in the overlap zone of partially trisomic individuals, a dicentric chromosome is formed, which is broken in anaphase II. This newly broken chromosome is healed by telomere formation. Here, we further test that model and show multiple examples of chromosome fission during meiosis followed by stable neotelomere formation. Nanopore adaptive sampling, a form of targeted sequencing, enabled us to enrich for neotelomere sites and characterize the sequence context of telomere formation. We find that neotelomere addition sites generally show at least 3-bp of microhomology to the telomerase RNA template. In most cases, perfect telomere arrays (TTTAGGG)n were added directly to identifiable sequence from the progenitor chromosome, although occasional insertions or unusual repeat structures were observed at the addition site. Nanopore sequencing also allowed us to measure cytosine methylation in the sequences directly flanking neotelomere formation sites. No consistent trend in gain or loss of DNA methylation was observed, but there were interesting exceptions where neotelomere addition appeared to cause changes in methylation over extended regions.

P188: ZYP1 mediates synaptonemal complex assembly and ensures crossover assurance in maize

Cytogenetics Tzu-Han Huang (Graduate Student)

Huang, Tzu-Han1 2
Wang, Chung-Ju Rachel2

1Institute of Plant Biology, National Taiwan University, Taipei, 106319, Taiwan
2Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 115201, Taiwan

During meiotic prophase I, homologous chromosomes undergo synapsis, mediated by the assembly of the synaptonemal complex (SC), a tripartite proteinaceous structure composed of two lateral elements (LEs) and a central region containing transverse filaments (TFs) and central elements (CEs). Although the SC is a conserved structure across eukaryotes, its regulatory roles in recombination remain unclear and somewhat controversial. In this study, we investigated the functions of ZYP1, the maize TF, to elucidate the role of synapsis in meiosis. In zyp1 mutants, despite the loss of the canonical SC formation, homologous chromosomes remained aligned at approximately 400 nm, connected by bridge-like structures coated with LE proteins. Most of these bridges were associated with nearby DMC1 filaments. In wild-type meiosis, ZYP1 rapidly polymerizes between aligned homologous chromosomes, with ZYP1 foci occasionally detected at these LE-derived bridges using super-resolution expansion microscopy, suggesting a temporal and spatial association between synapsis initiation and recombination engagement. Notably, the absence of ZYP1 modestly decreased crossover (CO) numbers. Although the CO numbers per cell in zyp1 mutants still exceeded the minimal requirement for holding ten chromosome pairs, defective CO formation resulted in some homologous chromosomes failing to form obligatory COs despite initially correct alignment, suggesting that ZYP1 is essential for CO assurance. Furthermore, we found that ZYP1 complexes modulate the loading of HEI10, a key CO regulator, independently of the presence of recombination intermediates. Together, we propose a model in which SC assembly promotes HEI10 condensation efficiency, thereby ensuring the formation of obligatory CO and achieving CO assurance.

Education & Outreach

P189: Digitizing maize cytogenetics history: Female meiosis and fertilization

Education & Outreach Taylor Isles (Graduate Student)

Isles, Taylor D1
Albert, Patrice S1
Birchler, James A1

1Division of Biological Sciences,University of Missouri, Columbia, MO 65211

Marcus Rhoades taught Cytogenetics beginning in 1940 at Columbia University, later at the University of Illinois, and finally at Indiana University from which he retired in1974. During the laboratory portion of this course, he used prepared slides of chromosomes illustrating numerous cytogenetic principles and phenomena. Some of these slides are those used in landmark publications in maize cytogenetics. In addition to contributions to this collection from members of his own lab, some slides were prepared by Barbara McClintock and Calvin Bridges among others. JB acquired this collection in 2012 from Ellen Dempsey who worked with Rhoades. We have begun digitizing this collection for its historical value and for educational purposes. The installment shown here display female meiosis and fertilization starting with a megaspore mother cell in the pachytene stage going through to the fertilization of polar nuclei by a sperm cell. Photographs of the illustrative cells and the narratives for each from Rhoades will be presented. Funding from NSF-MCB-2525949

P190: Maize genome editing and genetic transformation platform in Lyon, France

Education & Outreach Emilie Montes (Technician)

MONTES, Emilie1
ROGOWSKY, Peter1
WIDIEZ, Thomas1

1Laboratoire Reproduction et Développement des Plantes, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France.

Maize genetic transformation is a critical tool for functional genomics, cellular biology, and crop improvement. It enables the study of gene function, protein localization, and supports genome editing approaches like CRISPR/Cas for precise trait modifications. Established in 2008, our Maize Genome Editing and Genetic Transformation Platform is based on an Agrobacterium-mediated transformation protocol optimized for the A188 inbred line. The platform develops and implements innovative biotechnological tools to support research projects aimed at generating transgenic and genome-edited maize lines. In addition to supporting the team’s research on plant reproductive mechanisms (https://www.ens-lyon.fr/RDP/developpement-de-la-graine/), the platform is open to national and international collaborations. The platform’s skills are organized around three main areas: (1) Transformation tools: optimization of genetic transformation efficiency and development of new transformation methodologies. (2) Gene editing tools: CRISPR/Cas9 (PMID: 30684023) and base editing (PMID: 36518521) technologies. prime editing currently under development. (3) Genotyping and phenotyping tools: transgene insertion characterization, polymorphism genotyping, and rapid evaluation of new genome-editing tools in protoplast systems (PMID: 36518521). The platform also provides technical support and specialized training to researchers, students, and partners, including cloning, genotyping, maize transformation, and protoplast assays. Our public transformation platform ensures broad access to these technologies and sustains maize as both a major crop and a model species. More information is available on our web pages: https://www.ens-lyon.fr/RDP/developpement-de-la-graine/plateforme-de-biotechnologie-du-mais/

P191: Popcorn: The origin of maize and the future of science education

Education & Outreach Anthony Studer (Principal Investigator)

Shaffer, Marie N.1
Haas, Hannah E.2
Embry Mohr, Chris3
Studer, Anthony J.1

1Department of Crop Sciences, University of Illinois Urbana-Champaign; Urbana, IL, 61801, USA
2College of Education, University of Illinois Urbana-Champaign; Urbana, IL, 61801, USA
3Olympia High School, Standford, IL, 61774, USA

George Beadle proposed that teosinte was popped like popcorn prior to the selection of domestication alleles that exposed grain from beneath its hard seed coat. The keen observations and ingenuity of the ancient people of Mexico paved the path of improvement that has led to modern-day maize. Today, our team is using popcorn as an entry point to teach youth about domestication, plant breeding, genomics, gene editing, biotechnology, and data science. Not only is popcorn a universally recognizable and relatable agricultural product, but it is also a fun, healthy, and engaging snack available to all students. Our team delivers Authentic Research Experiences (AREs) to local high schools which give students an opportunity for hands-on participation in agricultural research. Students collect data and directly contribute to advancing crop improvement through their involvement in a popcorn breeding program tailored to their school. Currently, six schools participate in our AREs, half of which operate their own popcorn field site. Additionally, we are developing high school curriculum to equip teachers with comprehensive instructional tools that can engage and inspire large numbers of students. The Corn Next Generation Science Storyline curriculum, created as part of this project, has been piloted in over a dozen high schools, reaching well over 400 students. Once revised and reviewed, the curriculum will be made available on the United States National Science Teaching Association website where it can be accessed for free. Finally, we are also working with 4-H and United States Farm Bureau’s Ag In The Classroom to deliver themed agrotechnology content to younger ages. By working with schools and after-school programs, we hope to spark students’ innate curiosity that is vital to agricultural improvements of the future.

P192: Redesigning nitrogen flow in grass-dominated food systems

Education & Outreach Edward Buckler (Principal Investigator)

Buckler, Edward1

1Ithaca, New York, USA

Our modern food system reflects a combination of logical constraints imposed by energy and chemistry, alongside historical anomalies arising from evolutionary and anthropological pathways. Plants, particularly C₄ grasses such as maize, are exceptionally efficient at fixing carbon, yet they are energetically and chemically constrained in their ability to fix nitrogen. This mismatch led to a functional division in agriculture: biological systems fix carbon, while industrial processes now supply a substantial fraction of the world’s reactive nitrogen. The co-evolution of grasses, herbivores, and human ancestors produced a profound ecological transformation, one that continues to shape global diets. Today, human and livestock nutrition is dominated by grasses, which are rich in starch but relatively poor in high-quality protein. The Nitrogen 2.0 and CERCA projects seek to align agricultural practice with underlying biological and chemical logic by rethinking how synthetic nitrogen is allocated between cropping and livestock systems, including shifting a portion of nitrogen inputs directly to livestock rather than mostly through grain proteins and by promoting more circular cropping systems.

P193: The SI-MI mentoring program: A resource for guiding effective mentorship in the plant sciences community

Education & Outreach Brandi Sigmon (Steering Committee Member)

Sigmon, Brandi1
Puig-Lluch, Marcia2

1Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE
2ROOT & SHOOT Research Coordination Network, Rockville, MD

The ROOT & SHOOT Research Coordination Network consists of six plant science professional organizations who collaboratively design programming to address systemic barriers to full participation within our respective organizations. One focus area for ROOT & SHOOT is to develop resources to foster high quality, culturally responsive mentorship as plant scientists often have a high level of technical expertise due to their training, but need additional support in developing key relationship building skills for the effective mentorship of their mentees. Since supportive mentor-mentee relationships have been shown to be instrumental to the success and career persistence of the mentees, it is important to provide accessible and structured guidance for members of our professional organizations. To address this need, ROOT & SHOOT in collaboration with social science experts and members of our respective communities, have developed a free, cohort-based, online curriculum consisting of seven sessions to guide the development of effective, culturally responsive mentoring skills. This curriculum titled, Social Identity Matters in Mentoring (SI-MI Mentoring) Program, was designed as a series of interactive, engaging, and reflective workshops, which includes practice with impactful frameworks such as community cultural wealth, asset-based mentoring, social identity and intersectionality, and empathic conversations. The SI-MI Mentoring Program curriculum has recently completed evaluation by 17 beta-testers and following a final revision, will be made available as a free resource to the plant sciences community in 2026.

Quantitative Genetics & Breeding

P194: A dynamic genotype-environment integration approach decodes the genetic basis of phenotypic plasticity in maize

Quantitative Genetics & Breeding Pengfei Yin (Postdoc)

Yin, Pengfei1
Zhao, XiangYu1
Ji, Shenghui1
Guo, Jianghua1
Guo, Tingting2
Li, Kun1
Li, Weiya1
Xu, Gen1
Li, Xiaowei1
Zhang, Renyu1
Cai, Lichun1
Chen, Wenkang1
Fang, Hui1
Wang, Min1
Xiao, Yingni1
Yan, Jianbing2
Li, Jiansheng1 3
Yang, Xiaohong1 3 4
Li, Zhi5

1State Key Laboratory of Plant Environmental Resilience and National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China.
2National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
3Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, yinweoiChina.
4Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, China.
5State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Jinming avenue 1, Kaifeng 475004, China.

Under rapid climate change and increasing global food demand, enhancing crop environmental adaptability has emerged as a critical goal of intelligent breeding. Phenotype plasticity, driven by genotype-by-environment interactions (G×E), determines the capacity of crops to respond to dynamic conditions. Elucidating the genetic basis of these interactions is fundamental to understanding the regulation of phenotypic plasticity and developing climate-resilient crops. In this study, we collected flowering-time and the corresponding environmental data from 10 recombinant inbred line (RIL) populations across 12 planting environments and developed a proportional-time alignment model to investigate the dynamic G×E effects underlying flowering time variation. Our analysis revealed a photothermal time sensitivity window spanning 35% to 50% of the total sowing to flowering period, coinciding with key reproductive transitions such as female and male inflorescence development. Single-population linkage mapping detected 127 additive and 62 epistatic loci. Additive effects explained 10.2%-43.8% of the phenotypic variation, whereas epistasis accounted for less than 3%, indicating that additive effects play the predominant role in the environmental regulation of flowering time. Genome-wide association studies (GWAS) further identified 532 loci associated with photothermal time responses and pinpointed key flowering-time regulatory genes, including ZmMADS69, ZmELF3.1, CONZ1, ZmMADS1. These core genes, along with their epistatic interactions, are crucial for integrating environmental signals to modulate flowering time plasticity. This study establishes a unified framework for dissecting the genetic architecture of maize flowering-time plasticity, and provides a conceptual guidance for breeding climate-resilient crops with broad environmental adaptability.

P195: A research tool for the study of endosperm mutants in sweet corn (Zea mays L.)

Quantitative Genetics & Breeding Sophie Banks (Graduate Student)

Banks, Sophie K.1
Daza, Andy I.1
Tracy, William F.1

1Department of Plant and Agroecosystem Sciences, University of Wisconsin-Madison, Madison, WI, 53707

Sweet corn (Zea mays L.) exhibits a suite of genetic mutations that give rise to novel endosperm phenotypes. Included in these mutations are Sugary1 (su1), Sugary2 (su2), Shrunken2 (sh2), Dull1 (du1), and Waxy1 (wx1), all of which affect starch synthesis and are integral to perception of kernel flavor and quality. In the mid-1970’s, University of Wisconsin-Madison sweet corn breeder Dr. Robert H. Andrew developed seven different near-isogenic line (NIL) populations for the study of endosperm mutants and their effects on kernel sweetness. He used backcrossing to introgress the su2, sh2, du1, and wx1 mutations into seven different publicly available and commercially successful su1 sweet corn inbred backgrounds: C23, C40, C68, Ia453, Ia5125, IL101T, and P39, generating ten endosperm types for each inbred. Following Andrew’s work, Dr. Bill Tracy developed a second set of NILs exhibiting the same endosperm mutants in a starchy (Su1) background. The resulting populations have been used extensively as models for the investigation and characterization of endosperm carbohydrate composition, and more recently, for the assembly of a high-quality sweet corn reference genome. Here, we present a sampling of the extensive phenotypic diversity of these stocks, which will be made available to the public as a valuable teaching and research tool for studying the behavior of endosperm mutants in diverse commercial backgrounds.

P196: Adaptive plasticity of root system architecture as a response to Nitrogen availability drives yield gain over time in US maize hybrids

Quantitative Genetics & Breeding John McKay (Principal Investigator)

McKay, John K1
Mullen, Jack L1
Barnhart, Isaac2
Kelly, Courtland2
Clark, Randy2

1Soil and Crop Sciences, Colorado State University, Fort Collins, CO, 80523 USA
2Corteva AgriScience, Johnston, IA, 50310, USA

The steady increase in yield of US hybrid maize over the last 100 years an impressive achievement that is due to changes in both genetics and management. For the last 40 years, the management input of nitrogen has largely remained constant on a per acre basis, yet yields have continued the same rate of increase. Numerous studies have tested and found support for mechanisms that contribute to this continued genetic improvement of increased yield with the same level of N. These mechanisms include reduced N per unit mass of grain as well as increased N remobilization from stem and leaves. It has also been hypothesized that breeding for higher yield under constant N input has led to greater N uptake via roots, but data have been lacking to test this. We trialed Pioneer ERA hybrids across levels of the factor N fertilizer input and measured the response of yield as well as root system architecture. Our results support that selection on yield per se in research plots and farmers fields that have high heterogeneity in available N has led to increased plasticity in root system architecture as a function of N in newer hybrids as compared to older hybrids.

P197: Assembly and function of rhizosphere microbiome under drought stress during heterosis manifestation in maize

Quantitative Genetics & Breeding Ling Gu (Graduate Student)

Gu, Ling1 2 3
He, Xiaoming1 2 3
Huang, Xiaofang1 2
Li, Guoliang4
Tian, Tian1 2
Baer, Marcel1 2 3
Hochholdinger, Frank3
Yu, Peng1 2

1Plant Genetics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising 85354,Germany
2Emmy Noether Group Root Functional Biology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn 53117,Germany
3Crop Functional Genomics, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn 53117, Germany
4Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland 06466, Germany

Drought is a major abiotic stress threatening crop performance. Hybrids outperform inbred lines in productivity and stress tolerance. Root-associated microbes enhance plant growth and stress resilience, yet it remains unclear how heterosis influences the rhizosphere microbiome under abiotic stress and, in turn, how these microbes affect maize hybrids and inbred lines’ responses to drought. In this study, we planted 302 inbred lines from diverse genetic backgrounds and their F1 hybrids, generated by crossing each inbred with the common female inbred line B73, under drought and well-watered conditions. Overall, hybrids exhibited significantly higher shoot biomass than inbred lines under both water conditions, whereas mid-parent heterosis (MPH) for shoot biomass was markedly reduced under drought. Rhizosphere Shannon diversity was consistently lower in hybrids than inbreds, and drought significantly shifted rhizosphere community composition. Several bacterial genera were enriched in hybrid rhizospheres under drought and showed positive correlations with shoot biomass. Together, these results indicate that heterosis is associated with distinct microbiome assembly patterns, and drought modulates host–microbe relationships linked to growth. Ongoing work integrates microbiome features with host gene expression to identify mechanisms and inform breeding of drought-resilient maize.

P198: Bridging air and soil: Adaptive variation of maize brace roots in contrasting environments

Quantitative Genetics & Breeding Avani Vallayil (Graduate Student)

Vallayil, Avani1 2
Schneider, Hannah1 2

1Leibniz Institute of Plant Genetics and Crop Plant Research; Corrensstraße 3, Gatersleben, Saxony-Anhalt, Germany 06466
2Georg-August University Göttingen, Department of Crop Genetics, Göttingen, Germany 3705

The root system is fundamental to plant development and environmental adaptation, as it serves as the primary organ for absorbing water and nutrients from the soil. Among the various types of roots found in plants, brace roots are characteristic of monocotyledonous grass species (typically in the Paniceae and Andropogoneae), such as maize. In maize, brace roots play a crucial role by providing mechanical support to the plant and enhancing the uptake of water and nutrients. Originating from aboveground nodes, these roots differentiate into two types: those that remain aerial and those that enter the soil. Although this exposure to contrasting environments suggests divergent development, the specific structural and functional distinctions between aerial and subterranean brace root tissues are not well characterized. This project aims to investigate the anatomy, mechanical properties, hydraulic performance, and rhizosphere interactions of brace roots in maize. Comparative analyses will be conducted on brace roots from aerial and subterranean regions, assessing tissue organization, lignification, and vascular arrangement. Anatomical analysis suggests that brace root anatomy distinctly changes once the brace root enters the soil. Mechanical strength and water/nutrient transport will be measured. The integration of transcriptomic and metabolomic profiling will identify the molecular mechanisms underlying brace root specialization. Additionally, root anatomical modeling using tools like MECHA, GRANAR, and OpenSimRoot will provide insights into functional performance at different root tissues. Overall, this study will elucidate how brace root structural and functional diversity contributes to maize stability, resource acquisition, and adaptation to environmental variability.

P199: CERCA - Circular Economy that Reimagines Corn Agriculture

Quantitative Genetics & Breeding Cinta Romay (Principal Investigator)

Romay, Cinta1
CERCA, Initiative3
Buckler, Ed1 2 3

1Institute for Genomic Diversity, Cornell University, Ithaca, NY 14853, USA
2School of Integrative Plant Sciences, Plant Breeding and Genetics Section, Cornell University, Ithaca, New York, 14853, USA
3USDA-ARS, Ithaca, NY 14853, USA

Maize stands as the world’s most productive grain crop and a cornerstone of the global food supply. In industrial agriculture, it is primarily valued for its efficiency in starch production, with its protein content having relatively modest value. However, protein is the key driver behind most of the fertilizer demand, which constitutes the largest energy input for maize cultivation. The current combination of genetics and agronomic practices for high-yielding industrial corn leads to elevated costs for farmers, water pollution, and greenhouse gas emissions through the release of nitrous oxide. This poster highlights the progress of CERCA (Circular Economy that Reimagines Corn Agriculture), an initiative dedicated to enhancing maize’s photosynthetic and nitrogen efficiency while adapting the crop for modern applications and annual farm rotations. The CERCA team of over 90 scientists has already made remarkable progress. (1) Farm level modeling shows that a CERCA trait suite with –25% less fertilizer and –38% environmental impact, could still increase yield (+24%) (2) Conserved regulatory modules related to N recycling can be identified in perennial relatives of maize, providing candidate genes that can be leveraged (3) Reduction of N losses in the spring could be achieved by altering stover lignin content (4) N transport mechanisms in corn show natural variation that can be harnessed (5) The cob could be used for N storage (6) US landraces show potential as sources for cold and freezing tolerance (7) Comparative genomics points towards a shared and very rational way for plants to survive cold (8) Chilling tolerance and photosynthetic activity under cold can be improved by overexpression of selected candidate genes. The outcomes of this initiative will help develop nitrogen-efficient grain production that enhances farmer planting flexibility and reduces fertilizer inputs and environmental impact.

P200: Categorical and quantitative approaches for evaluating maize yield response to nitrogen

Quantitative Genetics & Breeding Donielle Brottlund (Graduate Student)

Brottlund, Donielle1
Thompson, Addie1
Lundquist, Peter1
Grotewold, Erich1

1Michigan State University; East Lansing, MI, 48824

Nitrogen (N) is one of the most costly and environmentally impactful inputs in maize production, yet hybrids differ substantially in their yield response and physiological sensitivity to N availability across environments. This variability complicates nitrogen management decisions, particularly as new hybrids are introduced under increasingly variable climatic conditions. While prior work has categorized hybrids into discrete nitrogen response classes, it remains unclear whether such classifications capture underlying biological mechanisms or adequately support prediction across environments.Here, we extend beyond descriptive classification to develop an interpretable, predictive framework for maize nitrogen response that integrates phenomic, agronomic, genetic, and environmental data. Using multi-year (2020–2024), multi-location field trials grown under contrasting N treatments, we quantify nitrogen responsiveness using yield-based and physiological response metrics derived from agronomic measurements. To link hybrid performance to genetic architecture, we incorporate both hybrids and their parental inbred lines and estimate general and specific combining ability (GCA, SCA) and genotype-by-environment (G×E) variance components using mixed models.Initial results indicate that additive genetic effects are unevenly distributed between parents, with male GCA contributing substantially more variance to kernel dry weight per plant than female GCA, while SCA remains a meaningful component of hybrid performance. Environmental effects and G×E interactions explain a large proportion of total variance, highlighting strong hybrid plasticity and year-specific responses. Building on these results, we are developing predictive models using Genomes to Fields data that evaluate the relative importance of parental genetics and weather variables in forecasting nitrogen response across environments.

P201: Combining canopy architecture and plot orientation to optimize light interception and distribution for improved maize grain yield

Quantitative Genetics & Breeding Gregory Schoenbaum (Research Scientist)

Schoenbaum, Gregory R.1
Alladassi, Boris M.E.1
Li, Dongdong1
Li, Xianran2
Yu, Jianming1

1Department of Agronomy, Iowa State University, Ames, IA 50011 USA
2USDA-ARS, Wheat Health, Genetics, and Quality Research, Pullman, WA 99164 USA

A century of maize breeding has produced hybrids capable of performing well at high populations, due in part to increasingly erect canopy architectures which improve light capture and overall photosynthetic efficiency. In recent decades, however, diminishing gains from erect leaf angles and elevated plant densities suggest both methods may have reached optimum levels. Considering this, we investigated whether the orientation of the maize canopy relative to incoming sunlight could be used to increase light capture and, therefore, grain production, and whether canopy structure was a factor. Using a split-plot experimental design, we evaluated twenty-eight maize testcross hybrids with diverse canopy architectures ranging from flat to upright throughout, including a variety of intermediates comprised of both flat and erect leaf angles. The experiment, performed in Ames, IA (USA), consisted of two replications of two-row plots planted either parallel or perpendicular to the general path of the sun. Field data were collected for population density, days to anthesis and silking, plant and ear heights, leaf angles at multiple canopy levels, and light interception at flowering. Post-harvest data collection included ear size measurements, grain moisture, and grain yield. Results indicate leaf angles and, importantly, their positions in the maize canopy are key determinants of light interception. Findings also show that canopy architectures allowing deeper penetration and, therefore, wider distribution of light throughout the canopy may promote overall plant performance by increasing the total number of leaves used for photosynthesis. This is especially beneficial in high-density scenarios where competition for limited light energy can be intense. Yield data and ear comparisons suggest plot orientation may have an effect on canopy performance and grain production. Identifying canopy architecture and plot orientation combinations to optimize light interception and distribution could enable producers to increase maize grain yield with minor adjustments to their current management strategies.

P202: Comparative analysis of U.S. ex-PVP and “Zemun Polje” commercial maize inbred lines using a 25K SNP array

Quantitative Genetics & Breeding Nikola Grcic (Research Scientist)

Ex-PVP germplasm are an important and useful resource for enriching established breeding programs, but its effective and successful implementation requires genetic analysis and the comparison with existing breeding material. In this study, we compared two panels of maize inbred lines genotyped using the same 25K SNP array: U.S. ex–Plant Variety Protection (ex-PVP) inbreds representing widely used elite germplasm, and commercial inbred lines developed in “Zemun Polje” maize research institute in Serbia. Using genome-wide SNP data, we examined population structure, genetic diversity, linkage disequilibrium (LD) and other parameters. Principal component analysis and model-based clustering revealed clear genetic separation between U.S. ex-PVP and “Zemun Polje” commercial inbred lines, with partial shared ancestry corresponding to major heterotic groups. Population structure analysis showed that “Zemun Polje” commercial inbred lines exhibited a higher proportion of Lancaster germplasm, whereas U.S. ex-PVP lines showed a greater contribution from the Iodent heterotic group. Within-group analyses indicated higher genetic diversity among Serbian inbred lines, consistent with their development from a combination of locally adapted and introduced germplasm from diverse sources over time. The obtained results provide an useful insight into the genetic background of analyzed material and will facilitate an effective use of ex-PVP germplasm.

P203: Cutting back on canopy: Tailoring plant architecture for the agronomic management strategies of tomorrow with a novel genetic modifier

Quantitative Genetics & Breeding Matthew Runyon (Graduate Student)

Runyon, Matthew J.1
Sible, Connor N.1
Arend, Miriam I.1
Li, Catherine H.1
Ghimire, Dinesh2
Robbins, Kelly R.2
Moose, Stephen P.1
Below, Fred E.1
Studer, Anthony J.1

1Department of Crop Sciences, University of Illinois Urbana-Champaign; Urbana, IL, 61801, USA
2School of Integrative Plant Science, Plant Breeding and Genetics Section, Cornell University; Ithaca, NY, 14853, USA

Increasing planting density has been a major catalyst for improved grain yield since the introduction of single-cross maize hybrids. Traits that have facilitated adaptation to elevated planting density by reducing interplant competition are often quantitative, resulting in modest rates of genetic gain over cycles of selection. As such, it is difficult to assess the impact of plant architecture changes on yield independent of other concurrently selected traits. To overcome this challenge, a single-locus mutation named reduced leaf area (rdla), which confers moderate reductions in total leaf area, was used to study the impact of leaf area on planting density tolerance. The rdla mutation was introgressed into a panel of 27 Expired Plant Variety Protection (ExPVP) inbred lines spanning heterotic subgroups, breeding eras, and commercial germplasm sources. Using a subset of these lines, a total of ten rdla and wild-type hybrid pairs were generated and grown at planting densities spanning 74,000 to 133,000 plants hectare -1 in two Illinois yield trial locations in the 2024 and 2025 field seasons. Manual leaf area measurements were collected on individual plants in both growing seasons, while aerial multispectral data were captured only in 2025 to differentiate rates of canopy closure across hybrids and densities. Leaf area reduction across the hybrid panel ranged from -25% to -45%, while yield reductions were less severe and did not follow a 1:1 correlation with leaf area. The yield gap between rdla and wild-type hybrids narrowed as planting density increased. The amount of grain produced per unit ear leaf area was significantly greater in rdla hybrids, suggesting an increased efficiency in grain production. Reduced total grain sink strength promoted higher nitrogen accumulation in stover, reducing grain nitrogen utilization efficiency. These results indicate that further optimization of leaf area and plant architecture using modifier traits like rdla is a viable approach toward designing an efficient crop canopy.

P204: Decomposing complex traits in maize without prior knowledge

Quantitative Genetics & Breeding Tingting Guo (Principal Investigator)

Guo, Tingting1 2
Yang, Haoyan1 2
Li, Xianran3 4
Yan, Jianbing1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China 430070
2Hubei Hongshan Laboratory, Wuhan, China 430070
3Department of Crop and Soil Sciences, Washington State University, Pullman, WA, USA 99164
4USDA-ARS Wheat Health, Genetics, and Quality Research Unit, Pullman, WA, USA 99164

Improving nitrogen use efficiency (NUE) is essential for sustainable agriculture, yet traditional and high-throughput phenotyping (HTP) traits have limited value as NUE proxies. We present a proof-of-concept study using artificial intelligence (AI) to autonomously characterize NUE. We analyzed 25,080 maize plant images under high-nitrogen (HN) and low-nitrogen (LN) conditions using a convolutional neural network (CNN). AI model learned 68 features, termed deep phenotypes. They achieved 96.7% accuracy in distinguishing N conditions versus 54.1% with HTP phenotypes. Genome-wide association studies (GWAS) revealed 523 nitrogen response-associated loci, a 25-fold increase over the 21 loci detected using HTP phenotypes. An interpretive framework linked AI discoveries to biological mechanisms. Field tests of 200 hybrids in varied N environments showed that each beneficial allele increased ear weight by an average of 18 grams per plot in LN conditions. Our findings provide new strategies for improving NUE and highlight the potential of AI in agricultural research.

P205: Designing interpretable AI models to identify drivers of maize phenotypic plasticity

Quantitative Genetics & Breeding Karlene Negus (Graduate Student)

Negus, Karlene L.1
Li, Xianran2
Welch, Stephen M.3
de los Campos, Gustavo4
Yu, Jianming1

1Department of Agronomy; Interdepartmental Genetics & Genomics, Iowa State University, Ames, IA 50011
2USDA-ARS, Wheat Health, Genetics & Quality Research, Pullman, WA 99164
3Department of Agronomy, Kansas State University, Manhattan, KS 66506
4Department of Plant, Soil and Microbial Sciences; Department of Epidemiology and Biostatistics; Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI, 48824

Artificial intelligence (AI) models excel at learning from data, but what can maize geneticists learn from AI models? Interpretable AI models remain challenging to design and have thus far received limited attention as a tool to improve our understanding about genotype-phenotype or environment-phenotype relationships. We explored the potential of using AI models to learn and identify the genomic regions and environmental factors that are most predictive of maize phenotypes. We developed a series of AI-genomic prediction (AI-GP) models with different design priorities featuring both simple (shallow-linear) and state-of-the-art (Jamba) AI architectures, and task layers designed to learn genotype-by-environment interactions. Using these models we evaluate the potential of AI-GP models for prediction of maize phenotypes and phenotypic plasticity in multi-environment contexts. We highlight two AI-GP models that allow us to compare the trade-offs of efficient and interpretable model designs. The efficient AI-GP model features fast computation and accommodates large genotype dataset sizes. In contrast, the interpretable AI-GP model enables identification of highly predictive genomic regions and environmental factor-genomic region combinations. Both models implement a novel prediction task-layer that identifies important environmental factors contributing to phenotypic plasticity making these models extensible to new environments with minimal impact on prediction accuracy. We validate these AI-GP models against conventional genomic prediction models using phenotypes evaluated in 11 environments from the maize Nested Association Mapping (NAM) population. Our results show that adapting AI model designs to be more interpretable for genomic prediction tasks – rather than simply merging existing AI and genomic prediction methods – enables the development of models with increased utility for maize genetics and breeding.

P207: Diverse defense responses of sorghum and corn to non-adapted rust pathogens

Quantitative Genetics & Breeding Libia Gomez Trejo (Graduate Student)

Gomez Trejo, Libia F.1 2
Mangal, Harshita2 3
Sigmon, Brandi3
Schnable, James C.2 3
Kim, Saet-Byul1 2

1Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
2Center for Plant Science Innovation, University of Nebraska-Lincoln, NE 68588, USA
3Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE 68588, USA

Maize and sorghum are globally important cereals with high synteny, yet both are threatened by fungal pathogens. Rust fungi can cause up to 40% yield loss in maize, while reports on sorghum rust disease remain limited. Over the past two decades, Rp1 alleles have been incorporated into maize inbred lines to confer resistance to common rust caused by Puccinia sorghi; however, this resistance was compromised by virulent isolates in the 1990s. Interestingly, common rust does not infect sorghum, despite its close genetic relationship to maize. We have found that 393 lines in the sorghum association panel display diverse defense-associated responses following inoculation with common rust, including distinct patterns of pigment accumulation and, in some cases, no visible symptoms. These responses have been categorized, and a GWAS has been conducted to identify candidate genes underlying these responses. In maize, 26 nested association mapping parent lines inoculated with the sorghum pathogen P. purpurea showed a hypersensitive response at pathogen entry points, suggesting active recognition and defense. Images of the development of the adapted and non-adapted pathogens both in maize and sorghum are shown for comparison. We plan to complement our current results with transcriptomic analyses to identify the genetic components responsible for the responses. This research enhances our understanding of how cereals interact with non-adapted rust pathogens and underscores the potential to incorporate new mechanisms into breeding strategies for durable disease resistance.

P208: Do we need dedicated maize varieties for perennial groundcover systems?

Quantitative Genetics & Breeding Memis Bilgici (Graduate Student)

Bilgici, Memis1
Lubberstedt, Thomas1

1Iowa State University, Ames, Iowa, USA, 50011

When maize is cultivated in perennial ground cover (PGC) systems, we face challenges related to competition between cash and cover crops for light, nutrients, and water. Understanding how different maize hybrids respond to these conditions, as well as the interactions between genotypes (G), environment (E), and management (M), is critical for optimizing yield and ensuring long-term sustainability. Our experiments address the question of whether dedicated breeding of maize hybrids suitable for PGC systems is needed. We screened 99 experimental hybrids (ISU) and 2 Corteva check hybrids to explore ranking differences for grain yield under conventional versus perennial ground cover (PGC) systems. Experiments were conducted with two perennial ground cover species (untreated (mowed)), Kentucky Bluegrass (KBG) and Poa bulbosa (PB), near Ames. The ranking of our hybrids for maize grain yield and plant height was substantially different under conventional and PGC conditions. However, our trials are very limited in the number of environments and, at best, preliminary. To develop maize suitable for PGC systems, breeders should prioritize selecting genotypes/hybrids based on performance in PGC environments rather than conventional ones, focusing on grain yield. Our KBG PGC system reflects the worst-case scenario: treatments to suppress the PGC failed, and corn competes with green PGC.In this situation, there would be a need to breed specifically for at least KBG systems, as performance in conventional environments may not reliably predict performance in “green PGC.” However, summer dormant and during summer “brown” PGC like PB (or treated KBG) appeared more promising and similar to conventional experiments.

P209: Drought tolerance classification using unmanned aerial systems with RGB and multispectral data

Quantitative Genetics & Breeding Juliana Yassitepe (Principal Investigator)

Duarte, Helcio1 2
Nonato, Juliana1 2
Duarte, Rafaela2 3
Gerhardt, Isabel1 2 4
Dante, Ricardo1 2 4
Yassitepe, Juliana1 2 4

1Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil, 13083-875
2Genomics for Climate Change Research Center, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil, 13083-875
3Embrapa Meio Ambiente, Jaguariuna, São Paulo, Brazil, 13820-000
4Embrapa Agricultura Digital, Campinas, São Paulo, Brazil, 13083-886

Field screenings for drought tolerance are costly and may be less accurate when relying solely on yield traits. To address this, breeding programs can adopt remote sensing technologies for quicker, more cost-effective assessments of how genotypes respond to drought. This study explores a drought tolerance classification system using vegetation indices derived from RGB and multispectral sensors, tested across eight machine learning models and four cross-validation scenarios commonly used in multi-environment breeding trials. Two trials—one under fully irrigated conditions and another under drought—were carried out in the 2023 off-season with maize hybrids. Morphological, flowering, and yield traits were used to derive drought coefficients and membership function values, which enabled classification of genotypes as tolerant or susceptible. Classification also utilized vegetation indices from sensors mounted on a drone that flew over the experiments during vegetative, flowering, and grain-filling stages. Each flight produced 35 (RGB) and 54 (multispectral) vegetation indices. The sensors and models within each cross-validation setup were evaluated based on accuracy, sensitivity, specificity, and F1 score. The training dataset—whether from irrigated or drought conditions—affected classification performance, particularly specificity. The best specificity, indicating fewer false positives, was achieved when models were trained on spectral data from drought trials. RGB data showed a slight edge over multispectral data, with an average improvement of 6.44%. The highest accuracy, F1 scores, and sensitivities were achieved with models such as k-nearest neighbors, random forests, and support vector machines with radial basis functions, applicable to both sensor types. Logistic regression performed well, but only with training data from irrigated trials. Conversely, the highest specificity values were observed with linear discriminant analysis and partial least squares across both sensor datasets. Support vector machine with a linear kernel also performed well in terms of specificity, especially in cross-validation scenarios involving untested environments. No single model outperformed others across all classification parameters. Additional experiments in 2024 are underway to broaden this study by including more scenarios across contrasting years.

P210: Dynamic calibration of maize growth model for enhanced decision-making in breeding and production

Quantitative Genetics & Breeding Xianlong Song (Graduate Student)

Song, Xianlong1 2
Li, Haitao1 2
Guo, Tingting1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China
2Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

Under global climate change, traditional maize growth models rely heavily on destructive sampling to calibrate cultivar parameters, resulting in low efficiency, high cost, and limited scalability for high-throughput breeding. To address these limitations, this study proposes a non-destructive dynamic calibration framework that integrates unmanned aerial vehicle (UAV) time-series remote sensing and machine learning to enable high-throughput estimation of cultivar parameters and support climate-smart breeding. The research follows an integrated “data–model–platform” framework. First, based on small-scale cultivar trials, multi-temporal UAV-derived vegetation indices (e.g., NDVI) were combined with ground-measured phenotypic data to develop a random forest model that establishes the relationship between remote sensing features and cultivar parameters, achieving non-destructive calibration. The framework was subsequently extended to the population level, where its cross-temporal and spatial generalization ability was systematically evaluated across multiple years, diverse regions, and contrasting nitrogen treatments. Genotype data were further incorporated to explore a genotype-based prediction pathway using GBLUP. Results showed that plant height, leaf area index, and biomass were more sensitive to growth-related parameters, whereas yield was primarily influenced by developmental parameters. UAV-predicted parameters exhibited strong agreement with those obtained using conventional methods (r > 0.75). The model achieved high accuracy in simulating biomass, leaf area index, and yield under cross-year and multi-nitrogen conditions (R² ≥ 0.82). The genotype-based prediction pathway demonstrated stable performance at the population scale, with simulation accuracy around 0.8 and a consistency index of approximately 0.9, confirming the feasibility of genotype-driven parameter prediction. By embedding genetic information into maize growth models, this study establishes an integrated intelligent breeding platform based on the closed loop of “data acquisition–model calibration–scenario simulation–breeding and production decision-making,” providing a scalable solution for climate-adaptive maize breeding and precision cultivation.

P211: Engineering maize root architecture via targeted genome editing of root regulatory genes

Quantitative Genetics & Breeding Marisa Miller (Research Scientist)

Mojica, Julius P1
Crawford, Brian CW1
Yurchenko, Olga1
Miller, Marisa E.1
St. Aubin, Brian1
Meister, Rob J.1
Deng, Molian2
Slewinski, Thomas L2
Lawit, Shai J1

1Pairwise, 807 E Main St., Durham, NC 27701, USA
2Bayer U.S. Crop Science, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA

Engineering root system architecture through precise genome editing offers a powerful route to improving crop performance under water- and nutrient-limited conditions. In this work, we describe a platform for targeted modification of potent genes for regulating root traits. Resulting allelic series produced heritable variation in root system architecture. These architectural changes are expected to enhance access to deep soil moisture and nutrients, providing a genetic lever to stabilize yield under variable environmental conditions. Together, this work establishes a broadly applicable, precise, and marker-free strategy to reprogram trait active gene targets, function and tune root traits in crops. Such targeted manipulation of root systems can complement conventional breeding and other genome-editing approaches aimed at above-ground traits, advancing the development of climate-resilient, high-yielding varieties.

P212: Engineering synthetic apomixis in maize

Quantitative Genetics & Breeding Nina Chumak (Postdoc)

Chumak, Nina1
Conner, Joann A.2
Ozias-Akins, Peggy2
Grossniklaus, Ueli1

1University of Zurich, Department of Plant and Microbial Biology, Zollikerstrasse 107, Zurich, CH-8008, Switzerland
2University of Georgia, Department of Horticulture, 2360 Rainwater Road, Tifton, GA 31793-5766, USA

Apomixis, asexual reproduction through seeds, is a reproductive strategy occurring in more than 400 species of flowering plants, but does not occur in the major crop species. Since apomictic plants reproduce clonally, their offspring is genetically identical to the mother plant. If engineered in crop species, apomixis would allow the maintenance of highly genetically heterozygous genotypes. For instance, it could be used to preserve the genotype of F1 hybrids over many generations without losing hybrid vigor. Current strategies to engineer apomixis require mutations in three to five meiotic genes causing a ‘mitosis instead of meiosis’ or MiMe phenotype, combined with the expression of a BABY BOOM-LIKE (BBML) transgene in the egg cell to initiated parthenogeissi. Although this strategy provided good results in rice, its implementation in current breeding schemes might be challenging due to requirement to manipulate multiple genes. Thus, simpler approaches to engineer apomixis may be advantageous.We performed genetic screens to identify mutations mimicking components of apomixis in maize and will present our progress in combining on such mutation with a BBML transgene to generate clonal seeds in maize.

P213: Evaluation of drought tolerance–related traits in native maize populations from the northeast of Mexico

Quantitative Genetics & Breeding Daniel Cruz Rosas (Graduate Student)

Cruz-Rosas, Daniel1
Mendoza-Gómez, Alejandro1
Rincón-Sánchez, Froylán1
Ruíz-Torres, Norma A2
Alonso-Nieves, Ana1

1Universidad Autónoma Agraria Antonio Narro (UAAAN), Plant Breeding Department, Buenavista, Saltillo, Coahuila, México. 25315.
2Universidad Autónoma Agraria Antonio Narro (UAAAN), Center for Seed Technology Training and Development, Buenavista, Saltillo, Coahuila, México. 25315.

Mexico is the center of origin, domestication, and diversification of maize, whose native varieties have adapted to diverse environments. These varieties maintain high levels of genetic diversity, allowing adaptation to diverse soil types, altitudes, and precipitation patterns. In Mexico, 59 native maize varieties have been identified, distributed throughout the country, including semiarid regions. In the state of Coahuila, in northeastern Mexico, eight native maize varieties have been reported and collected. In this region, annual precipitation is below the minimum required to complete the production cycle; therefore, most varieties have developed under some level of drought stress. This stress is considered one of the most damaging in agriculture, reducing corn yields by 15 to 25 %. For this reason, we aimed to characterize drought-tolerant native populations from northeastern Mexico. We evaluated 18 native populations under greenhouse and field conditions in 2025, inducing stress according to irrigation frequency. Preliminary results showed significant differences between drought levels and genotypes of the plant height, leaf area, and floral asynchrony. Plant height and leaf area in moderate drought decreased by 24 % and 37 %, respectively, with two populations of Cónico Norteño and one of Ratón standing out. According to the previous report, floral asynchrony in moderate drought increased by 52 %, pointing out two populations of Cónico Norteño and one of Celaya-Tuxpeño. We demonstrate that there is genetic variation among the populations from northeastern Mexico, which would be useful to generate improved populations for drought stress.

P214: Evaluation of morphological and reproductive traits associated with drought tolerance in native maize populations from the State of Coahuila, Mexico.

Quantitative Genetics & Breeding Alejandro Mendoza Gomez (Graduate Student)

Mendoza Gómez, Alejandro1
Cruz Rosas, Daniel1
Rincón Sánchez, Froylán1
Sánchez Ramírez, Francisco Javier1
Ruíz Torres, Norma2
Alonso Nieves, Ana Laura1

1Universidad Autónoma Agraria Antonio Narro (UAAAN), Plant Breeding Department, Buenavista, Saltillo, Coahuila, México. 25315
2Universidad Autónoma Agraria Antonio Narro (UAAAN), Center for Seed Technology Training and Development, Buenavista, Saltillo, Coahuila, México. 25315

Climate change has caused imbalances in environmental factors, such as temperature and precipitation patterns, thereby increasing the frequency and severity of droughts and adversely affecting plant development. In maize, water stress can significantly reduce yield, cause complete crop failure, and may even lead to the loss of local populations. In Mexico, locally cultivated native maize varieties have been selected over many years across diverse environments, resulting in well-adapted, location-specific populations that are valuable for germplasm selection and breeding programs. For this reason, 18 native maize populations from Coahuila were evaluated through greenhouse and field experiments in two locations. In the greenhouse evaluation, Ratón, Olotillo, and Cónico Norteño populations showed higher biomass accumulation under drought stress, likely due to physiological and morphological advantages associated with their origin in intermediate and high-altitude regions. Field evaluations showed significant differences in tassel length among populations, with Ratón and Cónico Norteño showing reduced tassel length under drought stress. These results suggest that the native maize populations from Coahuila harbor beneficial alleles for drought tolerance that could be integrated into the development of maize breeding programs.

P215: Expanding the use of biodiversity to safeguard future maize production

Quantitative Genetics & Breeding Ashish Saxena (Director)

Limited genetic diversity of elite maize germplasm raises concerns about the potential to breed for new challenges. Exploiting novel variation for climate-related stresses and genotype-specific root-soil interactions are important pathways to sustainably meet future maize food, feed and fuel security needs. When a species migrates from one ecosystem to another, changes in climate and geographical surroundings will result in adaptive changes of the allelic composition of the population. To identify and use novel genes from maize landraces, an environmental genome-wide association study approach (representing associations between SNP alleles and the original environment of accessions) was conducted. Almost 1000 landraces from 33 countries were selected. First-generation hybrids (F1) derived from landrace × elite crosses were tested under abiotic stress conditions and encouraging levels of adaptation and performance observed. Early testcrosses derived from S2 families were subsequently tested across three locations under heat and drought stress. These trials revealed useful genetic variation for yield and stress tolerance, indicating that landrace-derived genes can positively contribute to quantitative performance in breeding populations. Early stage of molecular validation by combining field performance data with DNA sequencing is currently underway. Results expected in 2026 will further support the role of landraces in building climate-resilient maize germplasm. Research suggests selection under high input environments has indirectly selected against genetic capability for sustainable nitrogen provisioning. Screening of elite tropical and sub-tropical advanced maize breeding lines for biological nitrogen inhibition (BNI) and root traits associated with rhizodeposition found associated traits to be highly polygenic. Key sources of sustainable nitrogen provisioning were primarily found in lines developed in breeding pipelines focused on low-nitrogen stress tolerance. These concurrent studies highlight the importance of conserving and using genetic resources to enhance the adaptive capacity of future maize varieties.

P216: From phenotypic tradition to genomic precision: Maize DUS 2.0

Quantitative Genetics & Breeding Anurag Daware (Research Scientist)

Daware, Anurag1
Hacke, Clemens1
Pécs, Márton2
Remay, Arnaud3
Starnberger, Philipp4
Schraml, Christina4
Schmid, Karl1

1Institute of Plant Breeding, Seed Science and Population Genetics, University of Hohenheim, Stuttgart, Germany
2National Food Chain Safety Office (NEBIH), Hungary
3Groupe d’Etude et de contrôle des Variétés Et des Semences (GEVES), France
4Austrian Agency for Health and Food Safety (AGES)

Plant variety registration requires Distinctness, Uniformity, and Stability (DUS) testing based on multi-year phenotypic evaluations that are time-consuming, costly, and sensitive to environmental variation. To improve efficiency, the International Union for the Protection of New Varieties of Plants (UPOV) has proposed three molecular marker-based integration frameworks (BMT Models 1–3). However, their application in maize remains limited due to a lack of validated marker–trait associations and robust predictive models. Using historical DUS phenotypic data from 352 European maize hybrids combined with high-density genotyping, we performed genome-wide association studies and identified 18 robust QTLs associated with 12 DUS characters, providing candidate genes for diagnostic markers development, relevant to BMT Model 1. Besides, we also developed an XGBoost-based genomic prediction framework for DUS notes, achieving predictive accuracy of up to 0.87 for selected characters (mean accuracy 0.67 across traits), exceeding performance reported in previous BMT Model 2 studies. Independent validation using more than 1,700 maize accessions from the USDA National Plant Germplasm System (USDA-NPGS) demonstrated cross-population transferability of both marker-based and prediction-based approaches, underscoring the value of public genomic resources for DUS applications. Despite inherent limitations associated with historical DUS datasets, our results provide evidence that molecular markers can enhance the efficiency of maize DUS testing, reducing time and cost without compromising decision reliability.

P217: Genetic analysis and gene cloning of maize waterlogging tolerant germplasm resource material HZ32

Quantitative Genetics & Breeding Xueqing Zheng (Graduate Student)

Zheng, Xueqing1
Qiu, Fazhan1

1National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, Hubei 430070, China

With the rapid changes in the global climate, waterlogging disasters have gradually become the main natural disasters affecting the growth of maize at seedlings. Therefore, identifying genes at the seedling stage of maize is of great significance for the genetic improvement. In this study, we filtered a maize backbone inbred line HZ32 with waterlogging tolerance and O167 with waterlogging sensitivity and we analyzed high-resolution transcriptome and proteome data of them after waterlogging at the seedling stage. It was found that the gene expression difference in HZ32 were relatively stable after waterlogging stress, and it may have the advantage of reserving relevant proteins to defend against waterlogging stress. Subsequently, the genomes of both HZ32 and O167 were sequenced, assembled and annotated. Through comparative genomics analysis with 14 reported genomes of maize inbred lines, it was found that both of them had rich genetic variations and HZ32 had more genes that might be involved in abiotic stress regulation. We conducted variation detection on HZ32 genome and O167 genome, and then combined the previous results of QTL related to waterlogging and the dominant genes screened by the transcriptome. Finally, we identify seven key genes that might be involved in regulating waterlogging tolerance at the seedling stage of maize. We verified the function of ZmRGLG1 among them. It may positively regulate waterlogging tolerance at the seedling stage of maize by regulating hormone homeostasis such as JA. Moreover, it was found this gene has protein interactions with multiple ERFVII subfamily genes that regulate the waterlogging tolerance of maize at seedlings, and it was promoted or inhibited by these ERFVII subfamily genes.

P218: Genetic and molecular analyses of European maize landrace for resistance to Fusarium stalk rot

Quantitative Genetics & Breeding Desmond Darko Asiedu (Graduate Student)

Asiedu, Desmond Darko1
Presterl, Thomas2
Kessel, Bettina2
Oyiga, Benedict2
Miedaner, Thomas1

1State Plant Breeding Institute, Universität Hohenheim, Stuttgart, 70599, Germany
2Kleinwanzlebener Saatzucht (KWS) KWS SAAT SE & Co. KGaA, Einbeck, 37574, Germany

Maize (Zea mays L.) is a globally important staple crop for human consumption and animal feed, with annual production reaching approximately 1.16 billion tons. However, maize productivity is severely limited by Fusarium diseases. Among these, Fusarium stalk rot (FSR) is the destructive soil-borne disease affecting maize-growing regions worldwide and poses a significant threat to sustainable crop production. FSR causes an average global yield loss of about 4.5%, with severe epidemics resulting in yield losses of 38 to 100% in the United States and Canada. FSR contaminates maize silage and grain with mycotoxins, creating serious risks for human and animal health. In Central Europe and North America, Fusarium graminearum is the predominant pathogen associated with maize stalk rot. Traditional methods including crop rotation and biocontrol agents have proven largely ineffective against FSR. Breeding for host resistance remains the most sustainable strategy for tackling FSR disease. European maize landraces (EML) represent a valuable source of quantitative resistance alleles and favourable agronomic traits, making them an important genetic resource for resistance breeding. This project aims to identify QTLs associated with FSR in a European flint maize landrace population using a genome-wide association study. Our first methodological study established needle injection as the most reliable inoculation method and introduced a robust quantitative phenotyping scale based on internode proportion, exhibiting high broad-sense heritability (H²=0.90). Our second study comprehensively reviewed the lifestyle of Fusarium pathogens and the genetic architecture of FSR resistance, identifying four major and numerous minor-effect QTLs, with a few candidate genes reported across only nine studies since 1993. Genomic selection was proposed as the most effective strategy for developing FSR resistance maize genotypes. Finally, we present two major FSR-resistant QTLs identified in the EML population ‘Kemater Landmais Gelb’ with direct relevance to resistance breeding.

P219: Genetic basis of leaf orientation responsiveness and yield stability in maize (Zea mays) under contrasting row spacing and cover crop management

Quantitative Genetics & Breeding Lakshmipriyanka Tammineni (Graduate Student)

Tammineni, Lakshmipriyanka1
Edwards, Jode1 2
Lübberstedt, Thomas1

1Department of Agronomy, Iowa State University, Ames, IA 50011, USA
2USDA-ARS Corn Insects and Crop Genetics Research Unit, Iowa State University, Ames, IA 50011, USA

Integrating cover crops into corn production is essential for soil health, but modern hybrids are often poorly adapted to the competition and light dynamics of alternative cropping systems. This research aimed to identify maize genotypes: “system specialists”, that maintain high yield and optimized leaf orientation in wide row spacing and cover crop environments. We evaluated 16 diverse inbred lines from the Germplasm Enhancement of Maize (GEM) project and their hybrid testcrosses. The experiment compared three management systems: standard narrow rows (control), wide rows without cover crops, and wide rows with cover crop (Tillage Radish). Our analysis revealed two critical findings. First, leaf orientation is a heritable, plastic trait. We found significant Genotype × Management interactions for leaf azimuth (p=0.0024) in inbred lines, meaning certain genotypes can physically re-orient their leaves to capture light in wider rows. Second, we identified a highly significant crossover interaction for grain yield (p

P220: Genetic dissection of the deep phenotype for nitrogen use efficiency in maize

Quantitative Genetics & Breeding Haoyan Yang (Graduate Student)

Yang, Haoyan1 2
Li, Xianran3 4
Yan, Jianbing1 2
Guo, Tingting1 2

1National Key Laboratory of Crop Genetic Improvement; Huazhong Agricultural University; Wuhan, Hubei, China 430070
2Hubei Hongshan Laboratory; Wuhan, Hubei, China 430070
3Department of Crop and Soil Sciences; Washington State University; Pullman, Washington, USA 99164
4USDA-ARS Wheat Health, Genetics, and Quality Research Unit; Pullman, Washington, USA 99164

Nitrogen use efficiency is essential to sustainable agriculture and global food security. However, understanding NUE is challenging due to its complex nature, influenced by genetic, environmental, and physiological factors. While high-throughput approaches have improved NUE phenotyping and genetic analysis, they still have limitations in accurately quantifying NUE and capturing its intricate patterns. Here, we conducted large-scale experiments under contrasting nitrogen applications and derived nuanced deep phenotypes to investigate genetic architecture of NUE. We collected 27,000 images from 420 pots under natural environments at three different growth stages and trained a deep learning model to classify the nitrogen conditions depicted in these images. The model achieved 95.2% accuracy in classifying nitrogen conditions. From these images, we extracted 68 deep phenotypes that were crucial in differentiating between high- and low-nitrogen conditions. Deep phenotypes exhibited larger coefficient variations and higher SNP-based heritability, compared to non-deep phenotypes. Particularly, we observed that certain deep phenotypes showed higher heritability under low-nitrogen conditions, in contrast to traditionally defined phenotypes that exhibited reduced heritability when nitrogen was limited. To uncover the genetic basis of NUE, we conducted genome-wide association studies using 990 phenotypes, including manually measured phenotypes, original high-throughput phenotypes, and deep phenotypes under both low- and high- nitrogen conditions, as well as combined nitrogen-response phenotypes. Our analysis identified 2,256 genes significantly associated with at least one phenotype, implicating genes such as nrt7, npf6, gst9, and myb25 involved in nitrogen uptake, transport, assimilation, and regulation. More than 95% of the identified candidate genes were associated with the deep phenotype. This integration of multi-environment trials and deep phenotyping methodologies effectively reveals potential NUE genes in maize and provides a framework for decoding important traits with genotype by environment interactions.

P221: Genetic diversity and identification of QTLs for low nitrogen in maize doubled haploid lines (DHLs)

Quantitative Genetics & Breeding Georgina Lala Ehemba (Postdoc)

Ehemba, Georgina1 2
Tongoona, Pangirayi2
Danquah, Erick Yirenkyie2
Ifie, Beatrice Elohor2
Das, Biswanath3

1Institut Senegalais de Recherches Agricoles, Hann Bel Air, Routes des hydrocarbures - BP: 3120 Dakar, Senegal
2West Africa Centre For Crop Improvement (WACCI), University of Ghana, PMB30, Legon
3International Maize and Wheat Improvement Centre (CIMMYT), Kenya,P.O. Box 1041-00621, Nairobi, Kenya

Drought and low nitrogen are stresses that strongly affect maize production in sub-Saharan Africa (SSA). The development of new inbred lines and the production of hybrids for drought and low nitrogen tolerance is key in addressing the food security problem in SSA. Therefore, the objectives of this study were to: i) assess the genetic diversity among newly developed DHLs, and identify candidate genes associated with low nitrogen (LN) tolerance in the DH lines. Two hundred and fifty doubled haploid lines were genotyped using mid-density genotyping with 3305 Single Nucleotide Polymorphisms (SNPs) to assess their diversity under low nitrogen and to identify candidate genes associated with LN tolerance. A high diversity was found among the DHLs with an average genetic distance of 0.38. Five clusters were found based on the Admixture and the discriminant analysis of principal components. Twelve SNPs for low nitrogen tolerance were also identified. The SNPs discovered were highly associated with the photosynthesis, biotic and abiotic stress tolerance. Key words: Low nitrogen, DHLs, SNP.

P222: Genetic mechanism analysis for spontaneous haploid genome doubling in maize

Quantitative Genetics & Breeding Wenhao Song (Graduate Student)

Song, Wenhao1 2
Qu, Yanzhi3
Kong, Zengguang2
Xiao, Yingjie1 2
Yan, Jianbing1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, Wuhan, 430070, China
3Yazhouwan National Laboratory, Sanya, 572024, China

Maize haploids exhibit high levels of sterility due to abnormal meiosis, but there is a phenomenon of spontaneous haploid genome doubling (SHGD) of haploids. However, the SHGD gene and genetic mechanism are unknown. We firstly generated F1 hybrids by crossing the maize inbred lines DX with B73, KN5585, Mo17, as well as RX with B73 and M119. Subsequently, these hybrids were subjected to parthenogenetic induction using the haploid inducer line YHI-1. We obtained haploid offspring (HF1) from 5 F1 populations and evaluated different levels of anther emergence and pollen production. The genotypic characteristics of the experimental materials were determined through whole-genome resequencing and molecular marker analysis. [Result] We obtained a total of 13 QTL associated with SHGD traits which distributed on seven chromosomes, chr1, chr3, chr4, chr5, chr7, chr8, and chr9. All identified QTL were validated via composite interval mapping, with 2 subjected to fine mapping through establishing segregating haploid progenies, where phenotypic metrics derive from progeny testing to link parental genotypes to offspring traits. Through cytological methods, we found that these special haploids (H-DX, H-RX) exhibit a highly similar meiotic process to diploids and are frequently present in critical stages such as dyad and tetrad cells. We constructed a complete ploidy map of the relevant materials by performing flow cytometry ploidy analysis on the relevant parts of the male spike during continuous development stages, and meanwhile we obtained corresponding RNA-seq data. The SHGD traits are controlled by multiple genes, these QTL can be used to assist the selection of high SHGD germplasm, and can improve the fertility recovery level of basic materials through polymerization, so as to promote the wide application of double haploid technology.

P223: Genetic pleiotropy in sorghum using a 503-trait meta-analysis spanning 18 years of studies

Quantitative Genetics & Breeding Harshita Mangal (Graduate Student)

Mangal, Harshita1
Linders, Kyle1
Jin, Hongyu1
Kuang, Xianyan2
Schnable, James C.1

1Department of Agronomy and Horticulture, University of Nebraska Lincoln, Nebraska, USA
2Natural Resources and Environmental Sciences, Alabama A&M University, Huntsville, Alabama, USA

Pleiotropy is a fundamental feature of genetic architecture, with human biobank studies demonstrating that a substantial fraction of genome-wide significant loci influence multiple traits. In plants, however, pleiotropy has traditionally been investigated through single-gene mutants rather than population-scale quantitative analyses, in part due to the lack of large, reusable multi-trait datasets. The Sorghum Association Panel (SAP) represents a unique exception: a community resource of genetically identical inbred lines repeatedly phenotyped across many years and environments, enabling the aggregation of otherwise independent studies into a unified multi-trait framework. Here, we compile and harmonize an expanded SAP trait resource, incorporating phenotypic data generated between 2008 and 2025 and increasing the total number of analyzed traits to 503. Using a meta-analytic approach, we leverage this extensive longitudinal dataset to characterize patterns of pleiotropy across diverse agronomic, developmental, and physiological traits without requiring all phenotypes to be measured within a single experiment. As a case study, we examine pleiotropic effects at the Dw1 locus, a major plant height gene on chromosome 9, and demonstrate that variation at this locus influences a broad suite of traits beyond height alone. Together, our results illustrate the power of long-term, community-generated datasets for dissecting pleiotropy in crop species and highlight how meta-analysis of reused association panels can reveal complex genetic relationships that are otherwise difficult to detect.

P224: Genome-wide response to divergent selection on vegetative phase change in maize

Quantitative Genetics & Breeding Heather Wodehouse (Graduate Student)

Wodehouse, Heather C1
De Leon, Natalia1
Tracy, William F1

1University of Wisconsin, Madison, WI, United States, 53706

Vegetative phase change (VPC) is the fundamental developmental transition from juvenile to adult vegetative tissue that is highly conserved across flowering plant species. VPC is regulated by a core genetic pathway wherein the miR156-SPL module acts upstream of the miR172-AP2 module. In maize, juvenile and adult leaves differ in epidermal and anatomical traits, including change in epicuticular wax, cuticle and cell wall thickness and composition, and cell types (e.g. trichomes and bulliform cells). Altering the timing of VPC impacts maize plant physiological maturity, plant architecture and susceptibility to pests. A recurrent selection experiment produced healthy maize populations with extreme early and late VPC, indicating the abundance of viable allelic variation, despite a limited range of VPC observed in standing maize diversity. This finding motivates our question: does selection act primarily on miR156 and miR172, on their targets the SPL and AP2-like families of transcription factors, on other established pathway components, or on previously uncharacterized loci? We used low-coverage WGS to sequence 96 individuals from each of three populations: an unselected base population cycle 19 early, and cycle 19 late. Then, because of the low read depth, we performed FST, Tajima’s D, and PCA selection scans based on genotype-likelihoods rather than hard genotype calls. These scans quantify genomic divergence between selection directions and the base population, identify candidate regions of selection, and assess their proximity to putative diverging components of the pathway.

P225: Genomic characterization of AAFC’s historical maize inbred lines and their application in GWAS

Quantitative Genetics & Breeding Aida Kebede (Principal Investigator)

Kebede, Aida Z1
Zhu, Xiaoyang1
Tenuta, Albert2
Belzile, Françoise3

1Ottawa Research and Development Center, AAFC, Ottawa, ON, Canada K1A 0C6
2Ontario Ministry of Agriculture, Food and Agribusiness, Chatham, ON, Canada N0P 2C0
3Université Laval Québec, QC, Canada G1V 0A6

Genetic diversity within breeding germplasm is fundamental for sustaining long-term genetic gain and responding to evolving breeding objectives. Agriculture and Agri-Food Canada (AAFC) has developed a unique collection of maize inbred lines since the 1920s, specifically adapted to early-maturity production zones in Canada. Despite their historical and breeding importance, this germplasm has not been fully characterized using modern high-throughput genotyping platforms. In addition its utility in genome-wide association studies (GWAS) has not been evaluated. In this study, we analyzed more than 500 AAFC historical maize inbred lines using genotyping-by-sequencing to generate a whole genome SNP dataset. These markers were used to quantify genetic diversity, assess population structure, define heterotic groupings, and conduct GWAS for flowering-time. The resulting insights provide a clearer understanding of the genetic architecture within this germplasm and highlight important loci influencing phenology and adaptation. This work enhances the strategic use of AAFC’s historical lines in maize breeding, enabling more informed parent selection, improved trait-mapping resolution, and stronger genomic selection training sets. The ultimate goal would be to support accelerated development of high-performing hybrids for short-season environments.

P226: Global genetic dissection of maize-teosinte divergence reveals EL3-2 as a pleiotropic domestication regulator

Quantitative Genetics & Breeding Xiaohong Yang (Principal Investigator)

Zhang, Renyu1
Zhang, Xuan1
Wu, Shenshen2
Cai, Lichun1
Li, Xiaowei1
Zhao, Xiaoyu1
Chen, Zhenyuan1
Chen, Wenkang1
Guo, Jianghua1
Li, Weiya1
Guo, Ce1
Yan, Dongzhe1
Li, Yangyang1
Luo, Yun2
Ge, Xingyu1
Li, Wenqing2
Zhuang, Junhong1 3
Yang, Fang2
Jackson, David2 5
Yan, Jianbing2 4
Li, Jiansheng1 3
Yang, Ning2 4
Yang, Xiaohong1 3

1Frontiers Science Center for Molecular Design Breeding, State Key Laboratory of Plant Environmental Resilience, and National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China
2National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
3Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, China
4Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China
5Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

Understanding the genetic basis of trait divergence between maize and teosinte is critical for accelerating maize improvement. Using a maize-teosinte (Mo17-mexicana) introgression population, we identified 93 additive quantitative trait loci (QTLs) and 9 pairs of epistatic interactions for 20 agronomic traits, revealing a polygenic architecture underlying the domestication and improvement of agronomic traits. The constructed QTL-trait network, together with identified genome-wide selection signals, revealed extensive genetic interconnections, with correlated traits sharing common loci, indicating a shared genetic basis underlying morphological divergence. Selection feature analysis further demonstrated that domestication and improvement, as well as mexicana introgression, acted on key genomic regions associated with agronomic traits. Furthermore, we cloned an ear length QTL, EL3-2, encoding a ULTRAPETALA (ULT) transcriptional regulator. EL3-2 functions as a central pleiotropic domestication regulator, modulating maize-teosinte divergence and regulating complex gene expression network across multiple developmental stages of maize. This study reveals the complex genetic basis of maize-teosinte morphological divergence and provides new insights that bridge evolutionary genomics with breeding practices for complex traits.

P227: How well do GWAS methods perform on TWAS data?

Quantitative Genetics & Breeding Marion Pitz (Postdoc)

Pitz, Marion1
Hochholdinger, Frank1
Lipka, Alexander E.2

1Crop Functional Genomics; Institute of Crop Science and Resource Conservation; University of Bonn; 53117 Bonn; Germany
2Department of Crop Sciences; University of Illinois at Urbana-Champaign; Urbana, IL 61801; United States

Identification of genes controlling quantitative traits is a key objective for not only maize genetics, but also for plant sciences and breeding. The increasing feasibility of large-scale expression studies led to the emergence of the transcriptome-wide association study (TWAS), which identifies statistical associations between gene expression and a trait. Although statistical models originally developed for the genome-wide association study (GWAS) are being employed for TWAS, their effectiveness in plant transcriptome data has not yet been benchmarked. Therefore, we evaluated the performance of TWAS on simulated traits that were controlled by gene transcripts in a manner analogous to genomic loci simulated to have quantitative genetic effects on a trait. Such gene transcripts, which we call Quantitative Trait expression Genes (QTeGs), were selected from transcriptome-wide gene expression data in four different maize populations. We then conducted a systematic evaluation of the ability of popular plant GWAS approaches to identify true and false positive TWAS associations. Our study demonstrated that GWAS models are useful for quantifying true positive statistical associations between QTeGs and traits. Nevertheless, we identified specific areas where improvements could be made. For instance, true positive identification of QTeGs was limited to those with the largest effects on the simulated trait. We also observed high variability in the number of false positive associations. Analogous to markers in linkage disequilibrium between genomic loci, these false positives putatively arise from their larger-than-average correlation with QTeGs. If follow-up studies confirm this inference, this finding should emphasize the importance of gene co-expression and network information in TWAS. Moreover, our results suggest that the inclusion of both expression-based relatedness matrices and multi-locus models enhance the reliability of TWAS results, primarily by decreasing the number of false positives. We hope that our observations will inform the selection and development of TWAS models as well as the interpretation of TWAS results.

P228: Identification and fine mapping of quantitative trait loci for anthesis silking interval in maize

Quantitative Genetics & Breeding Yapeng Wang (Postdoc)

Wang, Yapeng1
Liu, Siyu1
Chen, Chuan1
Yang, Shaoqi1
Zeng, Shirong1
Wang, Zhe1
Yang, Qin1

1College of Agronomy, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas/Key Laboratory of Maize Biology and Genetic Breeding in Arid Area of Northwest Region, Ministry of Agriculture, Yangling 712100, China

Abstract: Drought is a major environmental constraint that leads to a delay in silk emergence during the flowering stage, significantly reducing maize grain yield. Selection for a reduced anthesis to silking interval (ASI) has been demonstrated to be an effective strategy for enhancing genetic gains in yield under drought. An elite maize inbred line KB3020 showed short ASI under drought condition. Here, we identified two major QTL, qASI2.04 and qASI5.05, using an F2:3 population consisting of 286 families derived from a cross with KB3020 and HZ4. qASI2.04 explained 16% of the phenotypic variation, while qASI5.05 accounted for 14.3%. Phenotypic effects validation confirmed that alleles from KB3020 significantly reduced ASI at both loci. qASI2.04 was a partial dominant ASI QTL, while qASI5.05 was a recessive one. To fine map the two QTL, a large BC2F2 population was generated and screened for recombinants. Using recombinant-derived progeny testing strategy, we fine-mapped qASI2.04 to a 356-kb interval referring to HZ4 genome. Four annotated genes were predicted in this region. qASI5.05 was delimited to a 700-kb region and twelve candidate genes were identified. Using public RNA-seq data, we identified a tonoplast intrinsic protein gene within the qASI5.05 interval as the most promising candidate gene. Further studies will be conducted to validate its role by combining molecular, genetic, and physiological analyses. Collectively, the findings of this study will enhance our understanding of the genetic basis of ASI and facilitate marker assisted breeding for reduced ASI, a key trait for improving grain yield under drought stress.

P229: Impact of spontaneous haploid genome doubling on haploid induction in maize haploid inducers

Quantitative Genetics & Breeding Vencke Gruening (Graduate Student)

Grüning, Vencke K.1
Frei, Ursula K.1
Chen, Yu-Ru1
Lübberstedt, Thomas1

1Department of Agronomy, Iowa State University, Ames, Iowa, 50011, USA

Doubled haploid (DH) technology is a cornerstone of modern maize breeding, enabling the rapid development of homozygous inbred lines for hybrid production. This method significantly reduces the time required to develop inbred lines compared to traditional multi-generation self-pollination. A critical step in the DH process involves treating haploid seedlings with exogenous chemicals, such as colchicine, to induce genome doubling. However, this approach is costly, time-intensive, and involves toxic chemicals, necessitating careful precautions and additional labor. Spontaneous Haploid Genome Doubling (SHGD) offers a sustainable alternative by naturally inducing genome doubling, eliminating the need for chemical treatments, greenhouse space and the transplanting step. Integrating SHGD into DH inducer lines could reduce the cost and complexity of DH inducer line development. Developing inducer lines with increased haploid induction rates (HIR) will lead to more self-induced haploid plants, which are male sterile and will not shed pollen. Implementing SHGD into the DH inducer lines, will overcome this by restoring haploid male fertility (HMF). To evaluate whether HMF and HIR are independent traits, we developed a segregating haploid population from a cross between A427, which carries qshgd1 (associated with SHGD), and BHI306, which carries mtl (associated with HIR). The population was divided into two groups: one treated with colchicine and one untreated. Both treatment groups were transplanted to the field. In the haploid population (before pollination) 1000 plants were genotyped, and the remaining plants were genotyped in the doubled haploid population after successful pollination. Preliminary results confirmed a previously reported segregation distortion against mtl and showed a higher rate of successful pollination in plants carrying qshgd1. Moreover, preliminary results suggest no significant association between SHGD and HIR. These findings, though based on limited data, suggest potential for SHGD to facilitate advanced HIR performance haploid inducer improvement, since it leads to more self-induced haploid plants with restored male fertility. Additional genotyping and phenotyping of the doubled haploid population are currently underway and will provide improved insight into the combined effects of these traits.

P230: Insights into the biocontrol potential of Burkholderia gladioli B22 against maize stalk rot and promoting growth

Quantitative Genetics & Breeding Huanhuan Tai (Postdoc)

Tai, Huanhuan1
Wang, Tao1
Liu, Zhiyu1
Peng, Shanlin1
Liu, Haiyi1
Wang, Zhe1
Xue, Jiquan1
Yang, Qin1

1State Key Laboratory of Crop Stress Resistance and High-Efficiency Production/The Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest Region/College of Agronomy, Northwest A&F University, Yangling 712100, China

Maize stalk rot, caused by various pathogens including Fusarium graminearum (F.g), is a soil-borne fungal disease that seriously affects global maize production and quality. Developing beneficial rhizobacteria holds significant potential for disease control. In this study, we isolated Burkholderia gladioli B22 from the rhizosphere of disease-resistant maize and investigated its biocontrol potential and antimicrobial mechanisms. Strain B22 exhibited significant antagonistic activity against F.g and broad-spectrum antagonistic activity against various pathogenic fungi. Both crude extracts and volatile organic compounds (VOCs) from B22 effectively inhibited hyphal growth of F.g. B22 also displayed plant growth-promoting characteristics, including protease activity, siderophore production, phosphate solubilization, and nitrogen fixation. In pot trials, B22 inoculation significantly enhanced plant growth while reducing disease incidence of both maize stalk rot and wheat crown rot. Genomic analysis identified nonribosomal peptide synthetase (NRPS) gene clusters, suggesting a genetic basis for producing lipopeptide antibiotics. Metabolic profiling revealed that the VOCs produced by strain B22 primarily consist of dimethyl disulfide (DMDS), which has been demonstrated to disrupt the mycelial structure and inhibit the growth of F.g. These findings position strain B22 as a promising candidate for developing multifunctional biocontrol agents for integrated disease management and crop enhancement.

P231: Integrating genomic prediction and offset using DNA-pool genotyping to assess maize landrace adaptation and select accessions for pre-breeding

Quantitative Genetics & Breeding Stéphane Nicolas (Principal Investigator)

Nicolas, Stéphane D.1
Galaretto, Agustin O.1
Pégard, Marie2
Malvar, Rosana3
Moreau, Laurence1
Butrón, Ana3
Revilla, Pedro3
Madur, Delphine1
Combes, Valérie1
Balconi, Carlotta4
Bauland, Cyril1
Mendes-Moreira, Pedro5
Šarčević, Hrvoje6
Barata, Ana M.7
Murariu, Danela8
Schierscher-Viret, Beate9
Stringens, Alex10
Andjelkovic, Violeta11
Goritschnig, Sandra12
Gouesnard, Brigitte13
Charcosset, Alain1

1Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE - Le Moulon ; 12 route 128, F-91190, Gif-sur-Yvette, France
2INRAE, URP3F ; Le Chêne, Lusignan, France
3Misión Biológica de Galicia, CSIC; Pazo de Salcedo. Carballeira, 8 Salcedo, 36143, Pontevedra, Spain
4CREA; via Stezzano 24, 24126, Bergamo, Italy
5Agriculture School of the Polytechnic University of Coimbra (IPC-ESAC) and CERNAS – Research Centre for Natural Resources; 3045-601, Coimbra, Portugal
6University of Zagreb Faculty of Agriculture; Svetošimunska cesta 25, 10000, Zagreb, Croatia
7Banco Português de Germoplasma Vegetal, Quinta de S. José, S.Pedro de Merelim; 4700-8591, Braga, Portugal
8Suceava genebank; b-dul. 1 Mai 17, 720224 Suceava, Romania
9Agroscope; Route de Duillier 60, Case Postale 1012, 1260 Nyon 1, Schweiz, Switzerland
10DSP-Delley Semences et Plantes SA; Route de Portalban 40, 1567 Delley, Switzerland
11Maize Research Institute Zemun Polje, Belgrade, Serbia
12ECPGR, Alliance of Bioversity International and CIAT; Via di San Domenico 1, 00153 Roma, Italy
13AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro; F-34398, Montpellier, France

Maize traditional populations (landraces) hold valuable genetic diversity for addressing climate change and low-input agriculture but remain underutilized due to lack of evaluations. High-throughput pool genotyping (HPG) has been previously used to characterize diversity but its potential for implementing genomic prediction (GP) and genomic offset (GO) for maize landraces has not been tested yet. We developed HPG-based GP models—combined or not with GO and within-population diversity (Hs)—calibrated using 397 European landraces evaluated across environments and use cases. GP alone showed high predictive ability for yield (0.754), height (0.923) and male flowering time (0.943). Including Hs and GO in GP model improved yield GP of new landrace in new environment by 13%. Our model provided phenotypic adaptative landscapes for each landrace in future climatic scenario and predicted that agronomic performance stability increased with Hs. Combining GP with eco-genetic predictions can be used to identify promising genetic resources for adaptation to different target environments for prebreeding. p { margin-bottom: 0.25cm; line-height: 115%; background: transparent }

P232: Ion-know which genes control elemental traits!

Quantitative Genetics & Breeding Magdalena Janik (Graduate Student)

Janik, Magdalena1
Whitt, Lauren2
Ziegler, Greg2
Baxter, Ivan2
Dilkes, Brian3

1Washington University in Saint Louis
2Donald Danforth Plant Science Center
3Purdue University

Elemental homeostasis is one of the oldest problems life had to solve to secure its existence. Understanding how plants maintain specific elemental content (collectively referred to as the ionome) across orders of magnitude concentration variation in the environment is crucial to understanding plant physiology, plant environmental adaptation, and for plant uses. Plants are selective in their uptake, exclusion, and sequestration of elements which has profound effects on the planet we live in. The goal of this project is to develop, test, and disseminate to the community tools for plant ionome exploration. We have gathered published literature and curated a Known Ionomic Gene (KIG) list. This resource lists all genes known to regulate elemental accumulation. We expanded this resource to include orthologous genes in multiple species to improve gene annotations for these traits. To detect natural variation in the ionome we leveraged orthologous relationships to improve GWAS QTL detection and candidate gene identification for natural variation influencing the elemental contents of plant tissues. This combination of orthology and quantitative genetics has identified novel elemental genes as candidates and we are testing these to expand the genes known to affect elemental accumulation in plant tissues.

P233: K-mer based GWAS as a complementary approach to traditional GWAS across maize and sorghum genomes

Quantitative Genetics & Breeding Waqar Ali (Graduate Student)

Ali, Waqar1 2
Ullagaddi, Chidanand1 2
Turkus, Jon1 2
Schnable, James1 2

1Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, USA
2Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, USA

Traditional SNP-based GWAS relies on reference panels, which might potentially miss structural variants, presence/absence variation and copy number variation contributing to phenotypic variation. K-mers based GWAS is reference free approach that tests association between short DNA sequences and phenotypes directly from raw sequencing reads potentially capturing genetic variation invisible to traditional GWAS approaches. Here we describe an ongoing comparative study which implies both k-mer GWAS and traditional reference dependent GWAS across maize (545 genotypes) and Sorghum (938 genotypes) for a couple of traits including flowering time and disease resistance etc.Preliminary analysis of flowering time in maize and sorghum show promising signals with k-mer GWAS having peaks with improved detection. We hypothesize that traits influenced by presence/absence or copy number variation such as disease resistance like NLR genes might be better captured with k-mer approach compared to SNP approach. On the other hand, traits primarily governed by SNP type markers should show concordant signals between two methods a way to provide internal validation of k-mer approach.By running parallel both approaches, k-mer using presence/absence and traditional SNP based markers across couple of traits like flowering time and diseases resistance, we aim to establish scenarios where k-mer approach provides complementary or superior power to traditional approaches. This comparative phenomenon will help us to establish the scenario/protocol for when each method is most appropriate to use in crop genomics studies.

P234: Leveraging transcriptomic modulation to enhance genomic prediction of hybrid performance across environments

Quantitative Genetics & Breeding Jenifer Camila Godoy dos Santos (Postdoc)

Godoy dos Santos, Jenifer Camila1
B. Fernandes, Samuel2
N. Hirsch, Candy3
P. Berry, Sydney3
E. Lipka, Alexander1
O. Bohn, Martin1

1Department of Crop Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States
2Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, United States
3Department of Agronomy and Plant Genetics, University of Minnesota, Saint Paul, MN 55108, United States

Accurately predicting hybrid performance in variable and rapidly shifting environments remains one of the greatest challenges in maize breeding. Conventional genomic selection (GS) relies primarily on SNP markers, which capture only a portion of the biological processes underlying genotype-by-environment interactions (GEI). Despite plants dynamically modulating gene expression in response to abiotic stresses, this transcriptional plasticity has never been systematically incorporated into GS models for hybrid maize. To overcome this gap, our project aims to evaluate the potential of integrating transcriptome-derived features into GS models to predict the performance of untested genotypes across multi-environment trials. We have completed the first phase of the project by generating transcriptome profiles of both inbreds and hybrids grown under abiotic stresses. These data allowed us to quantify gene-level transcriptional modulation and to identify environmentally responsive co-expression networks. We are currently assessing strategies to incorporate these biologically informed features into genomic prediction pipelines using six multi-kernel GS models. A subset of these models, based on additive and non-additive genomic relationship matrices, has already been developed and serves as the baseline framework. The remaining models extend this framework by integrating transcriptome-derived features and, in some cases, environmental covariates to explicitly model GEI, thereby allowing a direct evaluation of potential gains in predictive accuracy relative to conventional genomic models. In the next phase, we will conduct stochastic simulations to explore practical strategies for integrating gene expression information into breeding pipelines. Overall, this work will enhance our understanding of how elite germplasm responds to environmental stress at the transcriptional level and provide biologically informed strategies to improve genomic prediction accuracy and accelerate the development of climate-resilient maize hybrids.

P235: Long-term selection reshapes the maize genome by elevating standing and rare beneficial variants to drive sustained gains in kernel oil content

Quantitative Genetics & Breeding Binghao Zhao (Postdoc)

Xu, Gen1
Zhao, Binghao1
Zhang, Xuan1
Wang, Min1
Du, Xiaoxia1
Fu, Xiuyi1
Feng, Haiying1
Yun, Tao1
Guo, Jianghua1
Sun, Fei1
Fang, Hui1
Li, Jiansheng1
Yang, Xiaohong1

1State Key Laboratory of Plant Environmental Resilience and National Maize Improvement Center of China, China Agricultural University, Beijing 100193, China

Long-term selection has enhanced the levels of oil content in maize kernels for decades, yet the genomic and evolutionary dynamics enabling sustained gains remains unclear. Here, we characterized two high-oil breeding populations, LDHO and KYHO, derived from diverse founders and advanced through multiple cycles of recurrent selection. Across 1,503 inbred lines encompassing eight selection cycles, oil content consistently increased in two populations without evidence of plateauing, despite largely stable genome-wide diversity. However, a marked rise in the deleterious allele burden in later cycles revealed the genetic cost of prolonged directional selection. Genome-wide scans identified 1,013 selective sweeps, often originating from rare beneficial alleles that steadily increased in frequency across selection cycles. These sweeps strongly overlapped with oil-associated loci that together explained ~50% of the phenotypic variation. Nearly half of the trait-associated loci fell within selection sweeps, directly linking selection signatures to functional variation. We further validated three positively selected genes that modulate oil accumulation, providing mechanistic evidence that connects genomic signals to trait biology. Our findings reveal a selection model where recurrent selection drives sustained improvement by exploiting standing and rare beneficial variation, concurrently increasing genetic load. This work provides a genomic and evolutionary framework for allele mining, genomic prediction, and the design of optimized breeding strategies in maize and beyond.

P236: Map-based cloning identifies two QTL genes for synergistic control of husk number in maize

Quantitative Genetics & Breeding Cuixia Chen (Principal Investigator)

Lu, Dusheng1
Xu, Xitong1
Wang, Yancui1
Wang, Shukai1
Han, Yanbin1
Chen, Cuixia1

1Shandong Agricultural Univ., Tai'an, Shandong, China, 271018

The maize husk provides essential protection for ear development, yet reducing husk number (HN) is a key breeding target for mechanized harvesting. As a complex quantitative trait, the genetic basis of HN remains poorly understood. Two major quantitative trait loci (QTLs) controlling HN: qHN1 and qHN6 were identified by using F₂ population and 10K SNP chip data. Map-based cloning delimited qHN1 to a 58-kb region containing a single gene encoding an AP2-family transcription factor, named ZmHN1. qHN6 was confined to a 1.2-kb region, with a GATA-family transcription factor gene, designated ZmHN6, located 1,712 bp downstream. Near-isogenic lines (NILs) for each QTL were developed through backcrossing and marker-assisted selection. Expression analysis revealed that ZmHN1 and ZmHN6 were significantly more highly expressed in the US043 (few-husk) background than in the BS351 (multiple-husk) background. Furthermore, loss-of-function mutants for ZmHN1 and ZmHN6, identified from an EMS mutagenized population, both exhibited a significant increase in HN. Combining published chromatin immunoprecipitation sequencing (ChIP-seq) data and dual-luciferase reporter (DLR) and electrophoretic mobility shift assays (EMSA), A DOF-family transcription factor demonstrated repressing ZmHN1 expression. The interaction between ZmHN1 and ZmHN6 proteins was confirmed by yeast two-hybrid (Y2H), GST pull-down, and luciferase complementation assays (LCA). The zmhn1/zmhn6 double mutant showed a more severe increase in HN than either single mutant, indicating their synergistic role in husk development. To evaluate the breeding value of these loci, the few-husk alleles of qHN1 and qHN6 from US043 were introgressed into the elite inbred line Chang7-2 via backcrossing and marker-assisted selection. The improved Chang7-2 showed a significant reduction in HN compared to the original Chang7-2. Moreover, the line pyramiding both alleles exhibited a stronger phenotypic effect than the single-allele introgression lines. In summary, this study identifies key genes regulating maize HN, unravels part of their molecular regulatory network, and provides valuable genetic resources and a theoretical foundation for molecular breeding of maize varieties suited to mechanized harvesting.

P237: Mining allelic diversity of maize landraces for abiotic and biotic stresses

Quantitative Genetics & Breeding Stéphane Nicolas (Principal Investigator)

Nicolas, Stéphane D.1
Balconi, Carlotta2
Butron, Ana3
Chaumont, François4
Gouesnard, Brigitte5
Frascarolli, Elisabetta6
Erdal, Sekip7
Esmeray, Mesut8
Lupini, Antonio9
Palaffre, Carine10
Faivre-Rampant, Patricia11
Bauland, Cyril1
Galaretto, Agustin O.1
Madur, Delphine1
Chapuis, Romain5
Torri, Alessio2
Mazinelli, Gianfranco2
Guyot, Joséphine4
De biasi, Mateo6
Hinsinger, Damien11
Redaelli, Rita2
Revilla, Pedro3
Draye, Xavier4
Malvar, Rosa Ana3
Moreau, Laurence1

1Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE - Le Moulon; 12 route 128, F-91190, Gif-sur-Yvette, France
2CREA - Research Centre for Cereal and Industrial Crops; via Stezzano 24, 24126 Bergamo, Italy
3Misión Biológica de Galicia, CSIC; Pazo de Salcedo. Carballeira, 8 Salcedo, 36143, Pontevedra, Spain
4Louvain Institute of Biomolecular Science and Technology, UCLouvain; 1348 Louvain-la-Neuve, Belgium
5AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, F-34398 Montpellier, France
6Department of Agricultural and Food Sciences, University of Bologna; Bologna, Italy
7Department of Field Crops, Batı Akdeniz Agricultural Research Institute; Antalya, 07100, Turkey
8Breeding and Genetics Department, Maize Research Institute; Sakarya, Turkey
9Mediterranea University of Reggio Calabria; Località Feo di Vito snc, 89122 Reggio Calabria, Italy
10Unité Expérimentale du Maïs, INRAe, Univ. Bordeaux; Saint Martin de Hinx, France
11Université Paris-Saclay, INRAE, Etude du Polymorphisme des Génomes Végétaux; Evry, France

Landraces are valuable sources of genetic diversity for addressing the challenges of climate change, reducing fertilizers and pesticides but remain underutilized in modern breeding programs. To fill this gap, the MineLandDiv project (Mining Allelic Diversity in Landraces for Tolerance to Abiotic and Biotic stress) aims at (i) identifying maize landraces and favorable alleles for tolerance to abiotic (heat/drought - cold - nitrogen) and biotic stresses (Corn borer) that could be used to broaden genetic diversity of modern breeding germplasms and (ii) better understanding their resilience to variable environmental conditions. We optimized a panel of 341 temperate maize landraces representative of European genetic diversity that we evaluated for roots architecture in platform and for various agronomic and physiological traits across a field trial European network of 6 locations using high troughput phenotyping / envirotyping tools. We observed a strong effect of stress for yield according to treatments and environments. We genotyped DNA pools of these landraces with 600 000 SNPs to conduct genome wide association studies with traits and environmental variables from landrace collecting sites. We identified genomic regions involved in trait and environmental variation. We developed a DNA pool targeted sequencing approach to compare haplotype contents at 14 genomic regions involved in tolerance to abiotic stresses using 372 landraces and 342 inbred lines. We showed that 17% of genetic diversity found in landraces has been lost in inbred lines. We developed genomic prediction that predicted with high accuracy agronomic traits in different environments. First results are encouraging to identify promising alleles and landraces for prebreeding.

P238: Modeling spatiotemporal variation in genotype-by-environment interactions of maize flowering time

Quantitative Genetics & Breeding Lorenzo Rocchetti (Postdoc)

Rocchetti, Lorenzo1
Desbiez-Piat, Arnaud1 2
Manceau, Loïc1
Wisser, Randall J.1 3

1LEPSE, INRAE, Institut Agro, 2 Place Pierre Viala, 34000 Montpellier, France
2Unité de Recherche Pluridisciplinaire Prairies et Plantes Fourragères, INRAE, 86600 Lusignan, France
3Department of Plant & Soil Sciences, University of Delaware, Newark, DE 19716, USA

Forecasting spatiotemporal patterns of genotype-by-environment interactions (GEI) remains a major challenge for designing crop breeding strategies, yet scalable frameworks are lacking. To address this challenge, we present a meta-model to virtualize the genetic transmission of polygenic variation (genome simulator) and developmental plasticity (crop growth model) leading to GEI for flowering time. We parametrize the genome simulator using empirical recombination rates and genome-wide marker effects (rrBLUP, BayesB) estimated for developmental traits measured in phenotyping platforms and field trials across a diverse panel of ~400 genotypes. Developmental variation predicted for virtual recombinants generated through genome simulation, combined with environmental conditions, enables the coupling of a crop growth model for simulating flowering time across large-scale virtual experiments, allowing an unprecedented dissection of GEI. Initialized from representative genotypes of maize diversity, we simulate thousands of virtual genotypes, analogous to those found in real breeding populations, and model their flowering times across thousands of environments encompassing extensive latitudinal variation together with past and future climate conditions. Using the Finlay–Wilkinson reaction norm framework to disentangle GEI, we gain mechanistic insight into the nature of variance scaling and genotype rank changes attributable to temperature and photoperiod responses. Our model highlights a key role of temperature on flowering time variance, suggesting a reduction in breeding progress in future environments.

P239: Molecular mechanism of maize qEL3.08 regulating trehalose metabolism and its impact on ear development

Quantitative Genetics & Breeding Aoqing Hu (Undergraduate Student)

Hu, Aoqing1
Qiu, Fazhan1

1National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, Hubei 430070, China

Maize is a globally important staple crop, and improving its yield plays a critical role in ensuring food security. The number of kernels per ear is a core trait determining corn yield, while ear length and kernel rows are key factors influencing kernel number. Therefore, understanding the genetic and biological mechanisms underlying ear length and kernel row formation is essential for increasing corn yield. Based on our previous findings, this project aims to further investigate the biological function and regulatory mechanisms of qEL3.08 in maize ear development using a range of genetic approaches. Additionally, we will use candidate gene association analysis to identify and select superior allelic variations of this gene and develop functional markers to support maize breeding. The project will also utilize advanced technologies such as single-cell transcriptome sequencing to explore the mechanism of qEL3.08 and its downstream target genes, further elucidating their roles in the genetic regulatory network governing maize ear development. We performed quantitative trait locus (QTL) mapping and successfully identified a major-effect QTL for ear length, qEL3.08, located on chromosome 3 of maize. The qEL3.08 gene encodes a transcription factor that regulates ear development by inhibiting the expression of ZmTPSs family genes, thereby modulating trehalose metabolism. The results will provide new insights into the role of trehalose signaling in maize ear development and offer theoretical and technical support for the breeding of high-yielding maize varieties.

P240: Multi-trait synergic selection by integrating prior genetic and environmental information in crop breeding

Quantitative Genetics & Breeding Wenjie Fang (Graduate Student)

Fang, Wenjie1
Yang, Wenyu1 2
Xiao, Yingjie1 2
Yan, Jianbing1 2

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2Hubei Hongshan Laboratory, 430070 Wuhan, China

With the continuous growth of the global population, limited arable land, and increasing challenges from climate change, achieving yield stability while meeting rising food demand has become a critical issue. Although numerous genes associated with agronomic traits have been identified, their direct application in breeding remains restricted due to the complexity of multi-trait inheritance and genotype-environment interactions. The objective of this study was to establish an improved prediction framework that integrates prior genetic and environmental information while explicitly accounting for multi-trait coordination, thereby enhancing the accuracy and efficiency of material selection in modern crop breeding. We developed the TOPlus model, which incorporates 160 cloned genes, detected QTLs, and 75 environmental variables. Using 5,820 F1 maize hybrids evaluated across five environments for 17 agronomic traits, we constructed an NCII-based training model and assessed its generalizability in independent AMP and commercial hybrid populations, to test robustness and transferability across diverse genetic backgrounds and ecological contexts. TOPlus achieved a mean yield prediction accuracy of 0.47 across five environments - improving over JGRA by 0.12 and over EADW+GW by 0.03 - and reached 0.66 on average for 16 additional traits. The model identified 11 candidate hybrids exhibiting high similarity to target varieties, superior yield potential, and temporal stability across environments. Gene contribution analysis further distinguished environmentally stable genes such as UB2 from environmentally plastic loci such as ZmTPS14.1, reflecting their distinct roles in developmental stability and stress responsiveness. Validation using 110 commercial hybrids across 14 field sites confirmed predictive capacity, with Denghai1810 correctly prioritized for the Huang-Huai-Hai region, consistent with its nationally approved production zone. By integrating genetic priors, environmental data, and multi-trait phenotypes, TOPlus provides a practical and scalable decision-support framework that enhances prediction accuracy, robustness, and material selection efficiency for the development of climate-resilient varieties.

P241: Multi-view BLUP: a promising solution for post-omics data integrative prediction

Quantitative Genetics & Breeding Bingjie Wu (Graduate Student)

Wu, Bingjie1
Xiong, Huijuan2
Zhuo, Lin3
Xiao, Yingjie1 3
Yan, Jianbing1 3
Yang, Wenyu1 2 3

1Hubei Hongshan Laboratory; Wuhan, Hubei, China 430070
2Huazhong Agricultural University; College of Informatics; Wuhan, Hubei, China 430070
3Huazhong Agricultural University; National Key Laboratory of Crop Genetic Improvement; Wuhan, Hubei, China 430070

Abstract: Phenotypic prediction is a promising strategy for accelerating plant breeding. Data from multiple sources (called multi-view data) can provide complementary information to characterize a biological object from various aspects. By integrating multi-view information into phenotypic prediction, a multi-view best linear unbiased prediction (MVBLUP) method is proposed. To measure the importance of multiple data views, the differential evolution algorithm with an early stopping mechanism is used, by which we obtain a multi-view kinship matrix and then incorporate it into the BLUP model for phenotypic prediction. To further illustrate the characteristics of MVBLUP, we perform the empirical experiments on four multi-view datasets in different crops. Compared to the single-view method, the prediction accuracy of the MVBLUP method has improved by 0.038–0.201 on average. The results demonstrate that MVBLUP is an effective integrative prediction method for multi-view data, which will accelerate crop breeding efficiency and facilitate future genomic design breeding for new varieties.Key words: Multi-view data, Best linear unbiased prediction, Similarity function, Phenotype prediction, Differential evolution algorithm

P242: Natural variation of liguleless1 promoter bound by ZmbHLH narrows maize leaf angle

Quantitative Genetics & Breeding Yaxin Wang (Postdoc)

Optimizing leaf angle (LA) to facilitate high-density planting is critical for increasing maize yield. LA is a complex quantitative trait controlled by multiple genes. While a few major LA regulators have been identified, the regulatory networks remain incomplete, and their utility is often limited by pleiotropic effects found in null mutants. Here, we exploited natural variation from a biparental population and clone the major QTL qLA2, corresponding to the promoter region of liguleless1 (lg1). Natural variation at this locus specifically modulates LA by fine-tuning lg1 expression without affecting tassel branch number. A bHLH transcription factor, ZmbHLH, whose binding affinity to the lg1 promoter is altered by the causal variant. ZmbHLH acts as a negative regulator of lg1, modulating leaf angle likely through the auxin signaling pathway. This study elucidates a novel regulatory module involving lg1 and its upstream transcriptional regulator, but also provides a precise genetic target for breeding compact maize varieties for high-density planting.

P243: Optimization of Diabrotica resistance in maize by enhanced nematode attraction

Quantitative Genetics & Breeding Niloufar Ashrafi (Graduate Student)

Ashrafi, Niloufar1
Schaff, Claudia1
Telleria Marloth, Janik1
Degenhardt, Jörg1

1Martin Luther University Halle-Wittenberg; Institute of Pharmacy, Department of Pharmaceutical Biotechnology; Halle, Germany 06120

ABSTRACT Maize cultivation is increasingly threatened by the Western Corn Rootworm (Diabrotica virgifera virgifera), a root-feeding pest continues to spread across Europe. Sustainable control strategies are urgently needed as chemical insecticides and crop rotation are becoming less effective. One promising natural defense relies on maize roots releasing a volatile signal, (E)-β-caryophyllene (EBC), which recruits beneficial nematodes that attack rootworm larvae. However, North American and many modern maize varieties have lost the ability to produce this signal, limiting the effectiveness of this putative biological control mechanism. The extent to which European maize hybrids retain functional variation for this trait remains unclear. Here, we investigated natural variation in volatile defense emissions among 12 European hybrid maize lines. We found pronounced differences in aboveground EBC emission, ranging from complete absence to high production, with several lines showing intermediate levels. These differences were accompanied by distinct emission profiles of other defense-related volatiles, indicating substantial diversity in indirect defense chemistry among European maize hybrids. Importantly, the presence of high EBC–emitting lines demonstrates that this potential defensive trait is often present in European genetic diversity. These findings reveal variation in maize indirect defenses which might be exploited for sustainable pest management. Ongoing analyses of root-induced EBC emission, gene expression, and allelic diversity will further clarify the genetic and regulatory mechanisms underlying this trait. Ultimately, this work might allow to breed maize varieties with enhanced resistance to western corn rootworm, reducing reliance on chemical insecticides and supporting environmentally friendly agriculture.

P244: Parsing genotype-interaction effects to understand the genetic architecture of maize cuticular wax accumulation across environments

Quantitative Genetics & Breeding Matthew Wendt (Graduate Student)

Wendt, Matthew M1
Hattery, Travis J1
Chen, Keting1
Granados-Nava, Karen1 2
Schroeder, Elly1 3
Myers, Bryn1 2
Moore, Riley1 4
Loneman, Derek1
Claussen, Reid1
Gilbert, Amanda1
Garfin, Jacob5
Bjerklie, Dane5
Lauter, Nick6 7
Hirsch, Candice N5
Yandeau-Nelson, Marna D1

1Dept. of Genetics, Development, and Cell Biology, Iowa State University; Ames, IA
2Undergraduate Genetics Major, College of Liberal Arts and Science, Iowa State University; Ames, IA
3Undergraduate Kinesiology Major, College of Human Sciences, Iowa State University; Ames, IA
4Undergraduate Biology Major, College of Liberal Arts and Sciences, Iowa State University; Ames, IA
5Dept. of Agronomy and Plant Genetics, University of Minnesota; Minneapolis, MN
6Department of Plant Pathology and Microbiology, Iowa State University; Ames, IA
7USDA-ARS Corn Insects and Crop Genetics Research Unit, Iowa State University; Ames, IA

The hydrophobic cuticle is comprised of a cutin matrix intercalated with cuticular waxes and plays a key role in protecting plants from drought and other environmental stresses. Maize silks, which facilitate pollination even under harsh environmental conditions, are unique due to their high accumulation of cuticular hydrocarbons. Both the relative abundance and total accumulation of cuticular waxes are impacted by genetic and environmental factors: modifying the cuticle’s efficiency as a hydrophobic barrier to non-stomatal water-loss. The genetic network that determines wax deposition and cuticle remodeling under environmental stress remains understudied, partially due to the difficulty in separating the environment-dependent and genotype-dependent components of genotype-by-environment variance. This genotype-by-environment decomposition traditionally necessitates plants be grown across many geographical locations and years. To address this, we implement a novel machine-learning pipeline to estimate genetic and genotype-by-environment variance from high-dimensional weather data for many cuticular wax traits simultaneously. We collected and quantified 45 wax metabolites from the silks of 448 inbred lines of the Wisconsin Diversity Panel (WiDiv), grown in two geographical locations (Iowa and Minnesota) across two years (2016-2017). Using multi-trait GWAS, we identify putative cuticular wax candidate genes explaining variation in maize silk wax accumulation across abiotic environmental gradients by estimating the contribution of alleles to both genotype and genotype-by-weather dependent wax variation. Additionally, we evaluate the importance of these loci for predicting wax accumulation for 12 WiDiv inbred lines grow across Iowa and Minnesota in 2020 and 2021. Characterization of genes that define the genetic architecture underlying cuticular wax accumulation will contribute to the design of weather resilient or “climate-smart” plants that remain healthy and productive under environmental stress.

P245: Phenotypic analysis of transgenic maize events under water stress conditions

Quantitative Genetics & Breeding Juliana Nonato (Postdoc)

Nonato, Juliana V A1
Bruno, Maria H F1 2
Riboldi, Lucas1
Pereira, Helcio D1
Duarte, Rafaela C R M1 4
Pinto, Maísa S1
Dante, Ricardo A1 3
Gerhardt, Isabel1 3
Yassitepe, Juliana E C T1 3

1Genomics for Climate Change Research Center (GCCRC), Universidade Estadual de Campinas, Campinas, SP, Brasil, 13083-875
2Departamento de Genética, Evolução, Microbiologia, e Imunologia- Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brasil, 13083-970
3Embrapa Agricultura Digital, Campinas, SP, Brasil, 13083-886
4Embrapa Meio Ambiente, Jaguariuna, SP, Brasil, 13918-110

Extreme climate events, such as drought and heat stress, directly affect maize physiological performance and yield. Plant responses to abiotic stresses are controlled by a complex network of gene interactions that can be modulated through various biological processes. Therefore, developing stress-tolerant cultivars has become a key focus in tackling the challenges posed by climate change. At the Center for Research in Genomics for Climate Change (GCCRC), we investigate uncharacterized genes that may be associated with drought tolerance, especially from extremophile plant species. Among these, we identified a promising candidate gene, designated Unknown 8 (UNK8). To evaluate its role in water deficit response, we tested three transgenic lines overexpressing this gene (UNK8_1, UNK8_2, and UNK8_3). The experiment was carried out in a controlled-environment growth chamber with a day/night temperature cycle of 25°C/22°C and a 16-hour light/8-hour dark photoperiod. Five replicates were used for each treatment, with the B104 inbred line acting as the non-transgenic control. Irrigation was stopped at the three-leaf stage (V3), and plants experienced drought stress for seven days, followed by rehydration. Plants were individually imaged on days 0, 3, 5, and 7 of stress and on day 10 after stress. Image-based phenotyping in R software was used to measure changes in plant height and leaf area over time. Additionally, fresh and dry biomass of shoots and roots was measured. All UNK8 transgenic lines performed better than the control, especially for traits related to biomass accumulation and leaf area maintenance under stress. The UNK8_3 line showed the strongest performance, with significantly higher values in most traits evaluated, except for plant height. In summary, our results suggest that UNK8 is a promising candidate gene for improving tolerance to abiotic stress and could help develop maize cultivars better suited to extreme climatic conditions.

P246: Phenotypic characterization of U.S. maize heirlooms

Quantitative Genetics & Breeding Melissa Draves (Graduate Student)

Draves, Melissa A1
Cummings, Jordan M2
Washburn, Jacob D3
Gage, Joseph L2 4
Holland, James2 4 5
Flint-Garcia, Sherry3

1University of Missouri; Division of Plant Science and Technology; Columbia, MO, USA 65211
2North Carolina State University; Department of Crop and Soil Sciences; Raleigh, NC, USA 27695
3USDA-ARS; Plant Genetics Research Unit; Columbia, MO, USA 65211
4NC Plant Sciences Initiative; Raleigh, NC, USA 27606
5USDA-ARS; Plant Science Research Unit; Raleigh, NC, USA 27695

Hybrid maize is the most productive crop in the United States, producing millions of bushels per year and is used for food, feed, and fuel production. Prior to maize hybrids, open-pollinated heirloom populations (landraces) were primarily used in the US for subsistence farming and livestock feed. Heirlooms are phenotypically and genotypically diverse, with many populations harboring unique alleles that are not found in modern germplasm due to the rapid selection for high yield that drove hybrid production. However, heirlooms are relevant in specialty markets as many chefs, organic farmers, and smallholder growers are interested in marketing heirlooms as a natural culinary product. Outside of the United States, heirlooms have been intensively studied, with extensive monographs written about populations from Mexico, South America, and Europe. However, there are no comprehensive datasets on extant heirlooms from the United States. This study aims to fully phenotype 990 heirlooms, primarily housed at the North Central Regional Plant Introduction Station. Heirlooms were planted in a partially replicated design in the summer of 2024 and 2025 in Columbia, MO and Clayton, NC. Manual phenotypic measurements including flowering time, leaf and tassel architecture, plant heights, ear disease, tillering data, and harvest traits were collected throughout the growing season. Preliminary cluster analysis based on phenotypes indicates known trends in historical heirloom data including the north to south pattern of geographical origin and unique phenotypes represented in Southwestern US germplasm. Ongoing efforts include an automated imaging pipeline to collect area, shape, and color measurements on ears, cobs, and kernels, and prediction of kernel composition traits using near infrared spectroscopy. Ultimately, this project will produce a comprehensive, public data set that will fully describe heirloom populations and their potential use for novel culinary traits.

P247: Plant–microorganism interactions as novel screening criteria in maize varietal registration

Quantitative Genetics & Breeding Anna Pia Maria Giulini (Research Scientist)

Novarina, Elena1
Bardelli, Tommaso1
Alali, Sumer1
Baccichet, Irina1
Nebuloni, Matteo1
Dal Prà, Mauro3
Zuffada, Mattia2
Alagna, Filippo2
Giulini, Anna Pia Maria1

1CREA Research Centre for Plant Protection and Certification, Via Venezian 22, Milan, Italy, 20133
2CREA Research Centre for Plant Protection and Certification, Via Emilia km 307, Tavazzano con Villavesco LO, Italy, 26838
3CREA Research Centre for Plant Protection and Certification, Via Guglielmo Marconi 2, Lonigo (VI), Italy, 36045

Climate change is increasingly recognized as a major driver shaping the epidemiology of plant diseases, with significant effects on agricultural productivity and global food security. Rising temperatures altered precipitation patterns, more frequent extreme weather events, and elevated atmospheric carbon dioxide (CO2) levels influence plant–pathogen interactions by affecting pathogen survival, virulence and spread, as well as host plant physiology and immune responses. Under current climatic scenarios, the ability of plants to actively recruit and interact with beneficial soil microorganisms is gaining recognition as a crucial component of plant resilience. Soil microbiota can enhance nutrient acquisition, stimulate plant defense mechanisms, and suppress soil-borne pathogens, which collectively reduce disease severity and dependence on chemical inputs. In this context, our research focuses on plant growth and its interaction with microorganisms with the final aim of identifying new traits to be considered during the varietal registration process of new maize hybrids prior to their release onto the market across Europe. In this study, we propose standardized basic methodologies to select plant genotypes more resistant to Aspergillus flavus and Fusarium verticillioides and that have the potential to recruit beneficial root-associated bacterial communities.

P248: Population-scale RNA sequencing reveals host genetic control of phyllosphere fungal communities

Quantitative Genetics & Breeding Charles Colvin (Undergraduate Student)

Colvin, Charles F1
Chopra, Surinder1

1Department of Plant Science; The Pennsylvania State University; University Park; Pennsylvania; United States; 16802

Fungal communities inhabiting aboveground plant tissues influence plant health, disease outcomes, and crop productivity, yet the degree to which host genetics shapes these phyllosphere mycobiomes remains unclear. Most prior studies have relied on amplicon sequencing and have identified few reproducible host–fungal associations, in part due to technical biases and an inability to distinguish active fungi from dormant or dead cells. Here, we demonstrate that standard polyA-enriched RNA sequencing of field-collected leaf tissue captures sufficient fungal transcripts to robustly profile metabolically active phyllosphere fungal communities, without targeted microbial enrichment. Leveraging population-scale RNA-seq datasets from maize, sorghum, and soybean grown across three field environments (2,194 total samples), we quantified hundreds of fungal taxa despite fungal reads comprising a small fraction of total sequences. This approach revealed extensive host genetic control over fungal community composition within each crop. Genome-wide association analyses identified numerous host loci associated with the abundance of individual fungal taxa, and transcriptome-wide association analyses uncovered coordinated host gene expression responses linked to fungal abundance, including defense-related pathways. As an illustrative case study, host loci associated with fungal abundance included a previously uncharacterized CC-NLR gene in sorghum exhibiting presence–absence variation, highlighting the ability of this framework to uncover biologically meaningful immune loci linked to specific fungal taxa. Together, these results demonstrate that host genetics exerts strong and reproducible control over active phyllosphere fungal communities across multiple crop species. Population-scale RNA sequencing provides a scalable framework for studying plant–fungal interactions and establishes a foundation for treating microbiome-associated traits as genetically tractable targets in crop genetics and breeding.

P249: Predicting biomass in drought-stressed young maize plants with machine learning using genomic and phenomic data

Quantitative Genetics & Breeding Domagoj Simic (Research Scientist)

Galić, Vlatko1
Vukadinović, Lovro1
Jambrović, Antun1
Šimić, Domagoj1

1Agricultural Institute Osijek; Juzno predgradje 17; HR-31000 Osijek, Croatia

Fresh and dry weight are important agronomic traits in young maize plants, which vary greatly with age, genotype, and growing conditions. Fresh weight (FW) includes all water, while dry weight (DW) is the solid plant matter (biomass) left after the removing of water. However, biomass prediction is still challenging, although there are a number of genomic and phenomic methods that generate large amounts of data. This study used phenomic data from chlorophyll fluorescence (ChlF) and hyperspectral imaging collected from a diversity panel of maize inbreds grown in a controlled environment and challenged by progressive drought stress. All inbreds were genotyped using the Illumina MaizeSNP50 array. The objective of this study was to compare predictions of FW and DW made by four machine learning (ML) algorithms based on a set of genomic and phenomic data in maize seedlings. Analysis was conducted starting with the loading of the dataset and selection of multiple measurement domains. The feature set included Q values from population structure analysis, spectral measurements from the ultraviolet-visible and near-infrared regions, ChlF parameters, calculated spectral indices, and several categorical variables. All data underwent standardization before model training. The four ML models were Partial Least Squares Regression (PLSR), Extreme Gradient Boosting (XGBoost), Random Forest, and a Convolutional Neural Network with a Multi-Layer Perceptron (CNN-MLP). CNN-MLP, the deep learning approach, achieved the highest accuracy, where R2 (Root Mean Squared Error – RMSE) was 0.81 (66.72 g) for FW and 0.74 (0.49 g) for DW. PLSR ranked second, where R2 (RMSE) was 0.64 (128.30 g) for FW and 0.61 (0.73 g) for DW. The integration of ChlF and hyperspectral data in combination with genomic data is recommended for predicting biomass in maize seedlings under drought-stressed environments. It could help improve management practices and contribute to the advancement of maize breeding and selection.

P250: Quantitative assessment of heterosis and combining ability for strategic hybrid breeding in maize for silage

Quantitative Genetics & Breeding Mythri Bikkasani (Graduate Student)

Bikkasani, Mythri1
Garg, Tosh1
Sandhu, Surinder1
Adapala, Gopikrishna2
BS, Vivek3
Vikal, Yogesh1

1Punjab Agricultural University, Ludhiana, Punjab, India, 141004
2International Crops Research Institute for the Semi-Arid Tropics, Patancheru, Hyderabad, Telangana, India, 502324
3International Maize and Wheat Improvement Center, Patancheru, Hyderabad, Telangana, India, 502324

Understanding combining ability and heterosis among diverse maize (Zea mays L.) germplasm is critical for improving hybrid performance for both grain yield and silage quality. In this study, 42 maize inbred lines representing tropical late-maturity, tropical early-maturity, temperate, and mixed tropical–temperate backgrounds were used to develop a multiple-hybrid population of 354 F₁ hybrids. Hybrids were generated using half-diallel and partial diallel designs within germplasm groups, along with North Carolina Design II (NCD-II) crosses involving tropical late × tropical early parents and tropical × temperate and mixed background parents. Parental lines and hybrids were evaluated in a single growing season across two locations for silage quality traits, including fiber components, crude protein, in vitro organic matter digestibility, and metabolizable energy. Significant genetic variation was observed among parents and hybrids for all traits, reflecting the broad genetic diversity of the germplasm. Estimates of general combining ability (GCA) identified inbred lines with consistent additive effects, while specific combining ability (SCA) revealed hybrid combinations with favorable non-additive interactions. Heterosis was evident for most traits and varied in magnitude across traits and environments, with tropical × temperate or mixed crosses generally showing higher average heterosis for grain yield components and energy-related silage traits than crosses within similar maturity groups. Kernel number–related traits contributed substantially to yield heterosis, while digestibility and energy traits exhibited notable hybrid vigor. Overall, the study demonstrates the potential of integrating diverse tropical and temperate maize germplasm to broaden the genetic base and enhance hybrid performance, providing useful insights for breeding strategies targeting simultaneous improvement of grain yield and silage quality.

P251: Rhizosheath inhabiting Massilia are linked to heterosis in roots of maize

Quantitative Genetics & Breeding Xiaoming He (Postdoc)

He, Xiaoming1 2 3
Gu, Ling1 2 3
Wang, Danning1
Baer, Marcel2 3
Hochholdinger, Frank3
Yu, Peng1 2

1Plant Genetics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
2Emmy Noether Group Root Functional Biology, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany
3Crop Functional Genomics, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany

Heterosis, or hybrid vigor, describes the superior performance of F1 hybrids compared to parental inbreds. While soil microbiomes are proposed to influence heterosis, it remains unclear how heterotic plants shape their microbiomes and how interactions relate to stress responses. Here, we investigate the role of rhizosheath formation—the soil tightly adhering to roots—in maize heterosis under nitrogen deprivation. Across sterilization, inoculation, and transplantation experiments, hybrids develop larger rhizosheaths than inbreds, and rhizosheath size associates with biomass heterosis. Rhizosheath-enriched genus Massilia correlates with lateral root density, rhizosheath size, and growth. Untargeted metabolomics and flavone-deficient mutants reveal links between Massilia and flavonoid pathways, while growth promotion by Massilia can also occur independently of host flavones. Metagenomic analysis shows that larger rhizosheaths recruit microbial functions related to nutrient cycling and stress adaptation. These findings identify rhizosheath formation as an integrative trait associated with heterosis and a promising target for breeding resilient crops.

P252: RoadMAPs to rhizosphere nitrogen retention: Breeding crops for microbiome-associated phenotypes

Quantitative Genetics & Breeding Angela Kent (Principal Investigator)

Kent, Angela D.1

1University of Illinois at Urbana-Champaign, Urbana, IL, USA 61801

Nitrogen losses from maize production reduce fertilizer efficiency and drive nitrous oxide (N2O) emissions through microbially mediated nitrification and denitrification. Although decades of breeding have improved yield and nitrogen use efficiency (NUE), selection has focused primarily on aboveground traits, with limited consideration of how plant genetics influence rhizosphere microbial functions that govern nitrogen retention.We approach key microbial nitrogen-cycling processes as microbiome-associated plant phenotypes that can be targeted through breeding. Our previous work used maize genotypes spanning domestication history to demonstrate that ancestral lineages related to teosinte exhibit significantly lower nitrification and denitrification rates compared to modern maize. These differences reflect shifts in microbial functional activity, suggesting genetic control over rhizosphere nitrogen transformations and a clear path toward breeding maize for reduced nitrogen loss.Using maize–teosinte near-isogenic lines, we identified genomic regions associated with two nitrogen-conserving traits: biological nitrification inhibition (BNI) and biological denitrification inhibition (BDI). Experimental hybrids carrying BNI alleles showed enhanced nitrogen retention without yield penalties, while BDI lines reduced N2O emissions. Both traits result in increased plant nitrogen accumulation. We envision a systems-based breeding perspective where we combine these traits with other NUE alleles and management BMPs.Building on these results, we are developing maize germplasm that integrates BNI, BDI, and complementary NUE traits to create NSave maize that conserves nitrogen in the rhizosphere while maintaining agronomic performance. Reintroducing microbiome-associated traits lost during domestication offers a pathway to improve NUE, reduce greenhouse gas emissions, and enhance the sustainability of maize production.

P253: Rooting for ecosystem services through haplotype analysis

Quantitative Genetics & Breeding Abby Livingston (Graduate Student)

Livingston, Abby1
Godoy dos Santo, Jenifer C.1
Ugarte, Carmen2
Bohn, Martin1

1Department of Crop Sciences, University of Illinois Urbana-Champaign
2Department of Natural Resources, University of Illinois Urbana-Champaign

Organic farming systems present a suite of biotic and abiotic challenges, including intense weed competition, pest pressure, and disease prevalence. Genotypic variation among maize cultivars plays a critical role in improving crop performance under these conditions. The root system, as the primary interface with the rhizosphere, has the potential to enhance ecosystem services important for productivity in organic systems. This project aims to identify root-associated haplotypes in maize that contribute to favorable rhizosphere functioning. We developed the Elite Maize Association Mapping Panel (EMAMP), consisting of over 1,650 recombinant inbred lines (RILs) derived from 66 elite F1 hybrids. All RILs were genotyped using the MaizeSNP50 array. Haplotype blocks will be extracted, converted into multiallelic markers, and used to define root haplotype clusters. RILs from the same haplotype cluster but with diverse genetic backgrounds will be crossed to generate hybrids. These inbreds and hybrids will be evaluated in organic field trials for root architecture and agronomic performance. Our goal is to identify root haplotypes that promote ecosystem services such as improved nutrient cycling, weed suppression, and stress resilience. This poster presents our genomic pipeline for haplotype identification and preliminary insights into haplotype frequency and potential function. Ultimately, this work supports the development of regionally adapted root ideotypes tailored to the needs of organic maize production.

P254: Screening of maize photosynthetic responses at low temperatures and high light

Quantitative Genetics & Breeding Emma Leary (Graduate Student)

Leary, Emma N1 3
McCubbin, Tyler J2 3
Washburn, Jacob D2 3

1Division of Plant Science and Technology, University of Missouri, Columbia, MO, USA 65211
2USDA-ARS Plant Genetics Research Unit, Columbia, MO, USA 65211
3Interdisciplinary Plant Group, University of Missouri, Columbia, MO, USA 65211

Nitrogen (N) availability in the soil accelerates in the spring as warming soil facilitates renewed microbial activity, whereas the demand for nitrogen by maize peaks later in the summer. This delay between peak nitrogen availability and plant uptake causes a substantial period of time during which soil N is not utilized for plant nutrition but is instead lost through N2O emissions, runoff, and leaching. Some of this N loss could be prevented by beginning the maize growing season earlier in the spring. Moving the growing season forward, however, means growing the plants at colder spring-time temperatures. This can be problematic for maize because its photosynthetic capacity declines under lower temperatures. Maize contains incredible genetic diversity and has been adapted to high elevations with low temperatures, offering promise for the existence of cold tolerant photosynthetic traits. Identifying these variations could improve modern maize and, along with frost tolerance, enable earlier planting. This project seeks to discover the genetic and physiological mechanisms that influence photosynthetic performance under cold conditions. We are screening for germplasm that exhibits healthy photosynthesis at cold temperatures across maize genotypes, landraces, and wild relatives. Controlled environment chambers are used to simulate a cold snap during the cool conditions of an early spring Midwest planting. A diverse collection of germplasm was assessed for responses to chilling stress. Variability of various aspects of photosynthesis and growth during chilling, including leaf elongation, chlorophyll content maintenance, as well as gas exchange fluorescence, suggest that there is a genetic component to sustained photosynthesis and growth during cold stress.

P255: Seeking the molecular drivers of hidden chilling susceptibility in maize

Quantitative Genetics & Breeding Catherine Giauffret (Principal Investigator)

Polvêche, Eugénie1
Lourgant, Kristelle3
Martin, Florence1 2
Roques, Sandrine1 2
Calatayud, Caroline1 2
Soriano, Alexandre1 2
Cornet, Denis1 2
Giauffret, Catherine1 3

1UMR AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France
2CIRAD, UMR AGAP Institut, F-34398 Montpellier, France
3UMR BioEcoAgro, AgroImpact, INRAE, Univ Liège, Univ Lille, Univ Picardie Jules Verne, F-80200 Péronne, France

Some maize inbred lines fail to survive after seed reserve depletion when exposed to temperatures below 13 °C for several weeks. Although this extreme chilling susceptibility is often masked in hybrid combinations, its early identification in newly introduced genetic resources and the elimination of major causative alleles from breeding material would substantially reduce efforts in breeding nurseries.We investigated the genetic and molecular drivers associated with a major quantitative trait locus (QTL) for chilling sensitivity repeatedly identified on chromosome 1 across several diversity panels and segregating populations. Heterogeneous inbred families were used to validate and refine the position of the causal region. A cold greenhouse experiment involving 930 segregating plants narrowed the interval to a 455 kb segment. Five families segregating for this region were subsequently phenotyped in growth chambers for approximately 30 traits.Multivariate analyses were applied to classify plants according to chilling symptoms severity. Twenty-eight contrasting individuals were selected for RNA-seq analysis. A Gaussian mixture model and a Random Uniform Forest algorithm consistently identified several clusters of chilling-sensitive plants, differing in symptom severity, but only a single cluster of tolerant individuals. At the transcriptomic level, sensitive clusters could not be distinguished, indicating a shared gene expression program among all sensitive plants. Comparison between sensitive and tolerant individuals within each family revealed 456 upregulated and 287 downregulated genes across the five families. None of the nine positional candidate genes (B73 AGPv5) were among these shared differentially expressed genes. Functional enrichment analysis highlighted approximately 40 downregulated genes related to transmembrane transport and about 20 upregulated genes involved in sugar metabolism.Our results indicate that long-term chilling sensitivity in maize is associated with a common metabolic reprogramming rather than differential expression of positional candidate genes, providing new perspectives for phenotyping strategies aimed at improving cold tolerance.

P256: Single-cell transcriptomics reveals cell-type-specific signatures of heterosis in early roots of maize

Quantitative Genetics & Breeding Danning Wang (Postdoc)

Wang, Danning1 2
Zhou, Yaping3
Chen, Xinping4
Hochholdinger, Frank3
Yu, Peng1

1Plant Genetics, School of Life Sciences, Technical University of Munich, Germany
2Plant Breeding, School of Life Sciences, Technical University of Munich, Germany
3Crop Functional Genomics, Institute of Crop Science and Resource Conservation, University of Bonn, Germany
4College of Resources and Environment, and Academy of Agricultural Sciences, Southwest University, Chongqing, China

Heterosis describes the enhanced growth and stress tolerance observed in hybrid plants relative to their parental lines. Crosses between genetically diverse inbred lines often result in longer primary roots, where enhanced root development plays a critical role in nutrient and water acquisition and strongly influences yield potential. Early root vigor drives rapid root system formation and is shaped by parental genetic diversity and the transcriptional regulation of key genes in maize. Single-cell technologies have been increasingly applied in plant research, providing unprecedented resolution to elucidate molecular mechanisms underlying responses to abiotic stresses. However, despite extensive studies of heterosis at the whole-organism and tissue levels, the cellular and molecular basis of heterosis at single-cell resolution remains largely unexplored. In this study, we investigated the cellular mechanisms underlying heterosis during early root development in maize by integrating single-cell RNA sequencing and spatial transcriptomics. To capture the genetic diversity of maize, the reference inbred line B73 was crossed with nine inbred lines representing three major heterotic groups: stiff-stalk, non–stiff-stalk, and tropical lines. Cell-level phenotyping revealed substantial variation in heterosis among hybrids, with crosses between B73 and non–stiff-stalk lines exhibiting significantly stronger heterotic effects compared to crosses from the other heterotic groups. By classifying genes into additive and non-additive expression patterns within each cell cluster, we observed a pronounced enrichment of non-additive genes in the outer root cell layers compared with inner layers, a pattern consistently detected in both single-cell and spatial transcriptomic datasets. Gene Ontology enrichment analysis further showed that these non-additive genes were predominantly associated with oxidative stress responses and related metabolic pathways. Overall, our findings provide the first single-cell and spatial transcriptomic evidence linking heterosis to specific cell layers during maize root development, highlighting the critical role of outer root tissues in driving heterotic vigor at the cellular level.

P257: Sources of transcriptomic variation in modern maize

Quantitative Genetics & Breeding Marcin Grzybowski (Principal Investigator)

Grzybowski, Marcin W.1
Schnable, James C.2

1Faculty of Biology, University of Warsaw, Warsaw, Poland, 02096
2Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, Nebraska, US, 68588

Genetic variation influencing gene expression plays a fundamental role in shaping phenotypic diversity. Despite its importance, large-scale studies of transcriptomic diversity in plants remain limited. Here, we analyzed gene expression profiles from 631 maize inbred lines using leaf tissue collected during a field experiment. We found that the vast majority of gene expression variation (95%) is distributed within maize heterotic groups (subpopulations), reflecting underlying DNA sequence diversity. We fine-mapped genetic variants associated with gene expression, cis expression quantitative trait loci (cis-eQTLs), and identified more than 64,000 putatively causal cis-eQTLs. A substantial proportion of genes (78.6%) harbored two or more independent cis-eQTLs, revealing extensive allelic heterogeneity in the genetic regulation of gene expression in maize. Finally, we observed that evolutionarily constrained genes tend to have fewer cis-eQTLs with smaller effect sizes, suggesting the presence of weak purifying selection against expression-altering variants. Together, our study provides new insights into the diversity and genetic regulation of gene expression in maize.

P258: Stable phenotypic shade-avoidance responses to low red/far-red ratio and shifting resource-use strategies in maize hybrids representing 100 years of long-term breeding for yield

Quantitative Genetics & Breeding Memis Bilgici (Graduate Student)

Bilgici, Memis1
Lubberstedt, Thomas1

1Iowa State University, Ames, Iowa, USA, 50011

The development of modern maize has led to significantly increased planting densities, yet the physiological basis for density tolerance remains a topic of intense debate. One prevailing hypothesis suggests that the selection for high-yielding varieties in high-density environments has reduced the shade avoidance response (SAR)—a set of phenotypic changes triggered by low red/far-red (R/FR) ratios—to avoid “costly” competitive traits related to plant architecture. Here, we evaluate the R/FR plasticity at the seedling stage of 27 maize hybrids released between 1920 and 2022. Our findings reveal that classic SAR characteristics, including increased plant height and reduced stem diameter, have remained remarkably consistent over a century of selective breeding. Importantly, the overall magnitude of the shade response—assessed through PCA-based trait shifts—shows no significant correlation with the year of hybrid release (Spearman rho = 0.098, p = 0.626). Rather than altering sensitivity to light quality, breeding efforts have led to a systematic reduction in leaf area, root length, and total stomatal pore area. These results indicate that maize density tolerance has been achieved through improved resource-use efficiency and structural compactness, rather than through the suppression of phytochrome-mediated shade signaling. Our findings suggest that R/FR-specific plasticity remains a valuable, untapped area for future molecular breeding aimed at enhancing canopy performance.

P259: Systems level plant-microbiome transcriptomics to explore maize and rhizosphere interactions underlying heat stress response

Quantitative Genetics & Breeding Nate Korth (Postdoc)

Korth, Nate1 2
Borrero, Isabella2 3
Rumley, Katelyn1 2
Woodley, Alex L.1 2
Choudoir, Mallory J.2 3
Gage, Joseph L.1 2

1Department of Crop and Soil Sciences, North Carolina State University, Raleigh, NC 27695
2NC Plant Sciences Initiative, North Carolina State University, Raleigh, NC, 27606
3Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695

Plant heat tolerance results from the combined effects of genotype, environment, the rhizosphere microbiome, and their interactions. Using a Genotype × Environment × Rhizosphere Microbiome (GERMs) model, we studied how plant and microbial gene expression profiles influence heat responses in maize and sorghum grown under control and heat-stressed conditions across two field soil treatments. We developed a systems-level metatranscriptomics approach to profile plant and microbial expression simultaneously and integrated these data with microbial community composition and plant performance. Plant and microbial gene expression was influenced by host identity, soil treatment, and temperature. Associations between microbial functions and plant heat tolerance phenotypes reflect coordinated, rather than independent, responses. Elastic net regression, random forest, and co-expression analyses identified groups of plant genes and microbial pathways consistently associated with heat tolerance, revealing cross-kingdom modules linked to signaling, redox balance, and carbon flux. A specific pattern was the enrichment of microbial D-amino acid metabolism, a poorly understood mechanism implicated in stress-mediated root–microbiome communication. Overall, these findings reveal that the rhizosphere microbiome actively participates in plant adaptation to high temperatures and, more broadly, to abiotic stress. Our systems-level approach emphasizes the value of the GERMs model for uncovering mechanistic plant–microbe interactions that could enhance crop resilience in a warming planet.

P260: Temporal 2D–3D phenotyping reveals dynamic maize architectural development across the growth cycle

Quantitative Genetics & Breeding Zhongjie Ji (Postdoc)

Ji, Zhongjie1
Ge, Yufeng1
Schnable, James1

1Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, 68505

High throughput phenotyping can be used to measure lots of stuff, but the high cost of conducting experiments with automated imaging means that data often collected either from only a period of development (frequently missing early vegetative phase and post flowering grain fill and drydown), or only for a few genotypes. Here we developed a mini-diversity panel of maize genotypes and collecting imaging data across nearly the entire maize lifecycle (15–90 days after planting). We employed a voxel carving algorithm to generate 3D plant reconstructions and voxel representations from multi-angle imaging (ten side views plus one top view) 2D images. From both 2D images and 3D voxels, we extracted a comprehensive suite of dynamic traits over time, including plant height, leaf number, voxel-derived geometric features, leaf shape parameters obtained through skeletonization and biomass partitioning. We found strong agreement between greenhouse and field measurements, with 3D-derived leaf angle in the greenhouse strongly correlated with field-measured leaf angle, and flowering time showing similarly strong cross-environment correlations. In addition, voxel-derived plant volume was highly correlated with destructively measured dry biomass of leaf and stem, indicating that voxel volume serves as a robust indicator of biomass accumulation. These analyses captured temporal variation and genotypic differentiation in key architectural traits throughout development. The resulting dataset provides a valuable resource for studying maize growth dynamics and offers a robust foundation for future research on plant phenotyping, genetics, and crop improvement. We also extended the voxel carving approach to Goodman–Buckler diversity which will enable quantitative genetics analysis such as GWAS, TWAS and eQTL.

P261: The Complementarity of Ex-PVP temperate and tropical lines in enhancing grain yield in tropical environments

Quantitative Genetics & Breeding Ashish Saxena (Director)

Low grain yield and frequent drought continue to hinder maize production in Sub-Saharan Africa (SSA), highlighting the urgent need for hybrids with better resilience. Utilizing ex-PVP (expired Plant Variety Protection) temperate maize lines is crucial for enhancing yield potential and genetic diversity in tropical-adapted varieties. This study involved crossing 15 temperate introgressed lines with 6 tropical lines and 8 single cross testers to produce 168 testcross hybrids, which were tested across 6 locations under well-watered (WW) conditions and 2 under drought-stressed (DS) conditions in Kenya. The analysis showed that temperate introgression generally improved yield performance across most tropical backgrounds. Hybrids from temperate introgressed lines outperformed hybrids developed from tropical lines by 1.20-2.04 t/ha under optimal conditions and yielded similarly under drought stress (1.88 t/ha for temperate introgressed lines versus 1.89 t/ha for tropical lines). Significant effects of genotype and site on grain yield (GY) and other agronomic traits, with pronounced genotype × environment interactions in drought conditions. Both general combining ability (GCA) and specific combining ability (SCA) were significant, showing additive variance was more influential in DS environments, while dominance effects were greater in WW conditions. In drought conditions, hybrids experienced a 74% reduction in GY, alongside 22% lower plant height (PH), 18% lower ear height (EH), and a 72% increase in anthesis-silking interval (ASI) compared to well-watered conditions. Lines L2, L10, and L16, along with testers T1, T2, T3, T4, and T7, demonstrated consistently positive GCA for GY and a shorter ASI, indicating their potential for breeding drought-tolerant, high-yield hybrids suited for SSA. These findings highlight the significance of ex-PVP lines as a resource that complements tropical lines, contributing to yield improvement in tropical rainfed environments. The interplay of additive and non-additive gene actions is crucial; leveraging pedigree methods alongside genomic selection for additive effects, combined with targeted heterosis, can expedite the creation of high-yielding, climate-resilient maize hybrids for stress-prone regions in SSA.

P262: The CorNBox, an effective platform for improving nitrogen utilization in U.S. corn belt maize

Quantitative Genetics & Breeding Steve Moose (Principal Investigator)

Moose, Stephen P1
Bubert, Jessica M1

1Department of Crop Sciences, University of Illinois at Urbana-Champaign, 61801

Maize grain yields in the U.S. Corn Belt have nearly doubled since 1980 despite little change in the amount of applied nitrogen (N) fertilizer or plant N uptake. The genetic basis for these gains in N utilization, the ratio of grain yield to accumulated plant N, are largely unknown. Although broad diversity exists for nitrogen utilization and its component traits, targeted breeding improvement has been hampered by phenotyping challenges and strong seasonal effects on yield responses to N. The “CorNBox” is a field site that combines management for a low and uniform soil N supply, detailed environmental sensing, and efficient methods for measuring nitrogen-responsive traits in maize. The CorNBox has been deployed for the past 15 years to screen maize germplasm and evaluate genetic gain for inbreds and hybrids with enhanced N utilization. The influence of genotype-by-environment interaction is moderated by growing in each season a reference population of 30 maize hybrids and their parents that represent the broader phenotypic diversity for nitrogen utilization. Results demonstrate that estimates of nitrogen utilization under N-deficiency have the highest heritability among NUE component traits, and that hybrids with superior NUE combine high N utilization and strong N-responsiveness. We document variability for N utilization among different testers, but selection for inbred parents with higher N utilization increases N utilization in derived hybrids regardless of tester. Crosses between selected high N utilization parents produces hybrids with better N utilization, but selection for N-responsiveness was less effective. All of the germplasm evaluated in the CorNBox has been genotyped, enabling biologically-informed genomic prediction from a dataset with thousands of phenotypic observations collected from tens of environments. Furthermore, comparisons of haplotype frequencies and gene expression in selected versus reference populations offers a complementary approach to ongoing functional genomics efforts for gene discovery.

P263: The PHENO_MaizE Project: UAV-based high-throughput phenotyping to support decision-making in maize breeding

Quantitative Genetics & Breeding Sofija Božinović (Principal Investigator)

Božinović, Sofija1
Pavlov, Jovan1
Grčić, Nikola1
Vančetović, Jelena1
Kovačević, Aleksandar1
Perić, Sanja1
Mladenović, Marko1
Lučev, Milica2
Galić, Vlatko3

1Breeding Department, Maize Research Institute Zemun Polje, Slobodana Bajica 1a, Belgrade, Serbia, 11185
2Research Department, Maize Research Institute Zemun Polje, Slobodana Bajica 1a, Belgrade, Serbia, 11185
3Maize Genetics and Breeding Department, Agriculture Institute Osijek, Južno predgrađe 17, Osijek, Croatia, 31000

UAV-based phenotyping enables the rapid collection of large volumes of data across numerous genotypes under field conditions and is increasingly recognized as a valuable tool in modern plant breeding. PHENO_MaizE is a three-year project funded by the Science Fund of the Republic of Serbia, aiming to explore the potential of RGB UAV imagery in maize breeding.Specifically, the project investigates how accurately digitally derived traits can replace conventionally measured traits, as well as their potential for predicting key agronomic traits such as flowering time, grain moisture at harvest, and grain yield. In addition, we aim to identify the most informative growth stages and digital variables that exhibit the highest predictive power.A total of 400 maize genotypes were selected for this study, including 200 parental lines (100 inbred lines and 100 doubled haploid lines) and their 200 test-crosses. During the 2026 growing season, these genotypes were imaged at two locations 22 times per location (44 UAV flights in total). RGB orthomosaics and digital surface models (DSMs) were generated for all flights to be used for per-plot digital data extraction. This experimental design enables the validation of prediction models under different testing scenarios, including known and unknown environments, as well as across different genetic materials (inbred lines and test-crosses).Furthermore, we developed a comprehensive and automated software platform for the efficient extraction, processing, and analysis of vegetation indices (VIs) from UAV imagery in maize breeding trials. In this poster, we present preliminary results from the first project year, along with an overview of future research directions.

P264: The combination of linkage mapping, genome-wide association study, and dynamic transcriptome analysis reveals conserved candidate genes for salt tolerance in maize

Quantitative Genetics & Breeding Ziyi Xiao (Graduate Student)

Xiao, Ziyi1
Pan, Zhenyuan2
Liu, Xinxin1
Zheng, Xueqing1
Wang, Guantao1
Fu, Yayu1
Li, Mengmeng1
Hou, Bin2
Li, Xuhua3
Zhang, Ming3
Jia, Chunlan3
Qiu, Fazhan1

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University / Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China
2College of Agriculture, Shihezi University / Key Laboratory of Oasis Ecological Agriculture Corps, Shihezi 832003, Xinjiang, China
3Shandong Denghai Seed Industry Co., Ltd, Laizhou 261448, Shandong, China

Salinization in China overlaps with maize-growing areas, severely affecting maize production. It is essential to uncover the genetic basis of salt tolerance and enhance maize’ s salt tolerance. Evaluated survival rates of a BC2F7 population and a natural population under salt stress in three environments. Performed QTL mapping and GWAS using genotyping data from 11312 SNPs in the BC2F7 and 3619762 SNPs in the natural population. Integrated QTL mapping, GWAS results, and dynamic transcriptome analysis to identify candidate genes. Identified three QTLs and 187 significant SNPs, including one QTL and 19 SNPs consistently detected across multiple environments. Six candidate genes were identified as potential contributors to salt tolerance. Few germplasms in the natural population possess multiple favorable alleles. This study expanded the understanding of the genetic basis of salt tolerance in maize at the seedling stage, providing critical molecular targets. Molecular marker-assisted selection could introgress favorable alleles into elite lines to enhance salt tolerance and facilitate yield increase in saline-alkali soils.

P265: The effect of selection on allele frequencies and diversity in wheat (Triticum aestivum)

Quantitative Genetics & Breeding Mirai Inaoka (Graduate Student)

Inaoka, Mirai1
Sneller, Clay1

1Ohio State University, Wooster, OH, USA 44691

Soft red winter wheat (Triticum aestivum L.) is traditionally improved by selecting lines with the best trait values and then intercrossing them through phenotypic selection (PS). A newer approach known as genomic selection (GS) utilizes individual DNA fingerprints to predict the trait values of lines and using them to determine the best lines to advance. However, there is limited information on the effect of PS or GS on genetic diversity. The objectives of this study were to assess changes in allele frequencies due to breeding and determine if the changes were due to random drift or selection. Allele frequency changes were assessed for 1,173 markers in 4,678 OSU lines from ten cohorts as they were advanced through trait evaluation stages 1 to 4. By simulating the allele frequency changes occurring by random chance, it was possible to determine the probability of an observed change occurring by drift alone. 5.54% of markers had large directional changes with an absolute frequency change greater than 0.15 in 33% of selection scenarios. The greatest change occurred as lines were advanced from stage 3 to 4, which resulted in fixation in 24.1% of the selection scenarios. As much of the change occurred during the final stage of advancement, negative impacts of the change could be mitigated by selecting parents earlier in the pipeline or using selection criteria that considers diversity. This study documented genome changes that occur during breeding, thus enabling plant breeders to make informed decisions regarding managing the diversity of their breeding populations.

P266: The genetic architecture of acyl-lipid traits in maize kernel and leaf

Quantitative Genetics & Breeding Hui Li (Principal Investigator)

Pei, Laming1
Zhu, Jiantang1
Yang, Yaqing1
Duan, Wencheng1
Huang, Mengyue1
Wen, Weiwei2
Yan, Jianbing2
Li, Hui1

1School of Biological Science and Technology,University of Jinan, Jinan, China
2National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China

Lipids, as important branch of metabolites, are one of the major components of bio-membranes, which delineate the interface between cell and environment and divide cells into different compartments. In addition, lipid participates in the necessary life activities, including carbon storage, energy conversion, and signal transduction. In this study, we sought to identify the genetic determinants of 249 acyl-lipid species, measured using ultra-performance liquid chromatography coupled with Fourier transform mass spectrometry (UPLC-FT-MS) on immature kernel, mature kernel and mature leaf from 513 diverse maize inbred lines. To uncover genetic control of the lipid metabolic traits, we conducted lipid-based GWAS (LWAS) of 495 acyl-lipid traits using the Q+K mixed model in each group. In LWAS, more than 36.6% (181/495) of the acyl-lipid species had at least one association locus at a genome-wide significant level P ≤ 2.04 × 10-6. Totally, 290 polymorphism-trait associations were identified in three groups, resulting in, on average, around two associated loci for each acyl-lipid metabolite feature. Each locus explained 5% - 29% of the observed lipidic variance, with a median of 10.0%, 5.7% and 6.4% for IMK, MK and ML, respectively. By performing LD (linkage disequilibrium) analysis of significant SNPs on the same chromosome, a total of 37, 81 and 73 unique significant associations for IMK, MK and ML, respectively, were identified. The 159 candidate genes were significantly enriched in GO terms lipid metabolism and associated processes. We next investigated the association between the variants and the expression levels of the loci identified by LWAS. Expression data were available for 95 of the 159 candidate genes from IMK and ML RNA-seq datasets of 508 association mapping population. Besides, 34 (21.1%) genes were associated with more than one acyl-lipid traits at P ≤ 2.04 × 10-6. Among 34 pleiotropic genes, only six genes associated with different tissue’s lipid metabolites, indicating discrepancy of lipid metabolism in kernel and leaf. This study elucidates the genetic architecture of lipid metabolism in maize, identifying key loci and tissue-specific regulatory genes with pleiotropic effects, which provides a foundation for understanding lipid functions and breeding for improved traits.

P267: Understanding the morpho-physiological framework of ear flex for improving yield and yield stability in maize

Quantitative Genetics & Breeding Yuki Matsumura (Graduate Student)

Matsumura, Yuki1
Yamamoto, Haruho2
Kon, Akihito3
Murai, Yusuke4
Horikoshi, Mizuki1
Harasawa, Yuri1
Hirota, Kazuya1
Hiyama, Kaishi1
Ikegami, Hiroki2
Uchida, Kentaro2
Kashiwagi, Junichi1
Ichikawa, Shinji5
Nakashima, Taiken1

1Graduate school of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan
2Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan
3Animal Research Center, Hokkaido Research Organization, Shintoku, Hokkaido, Japan
4KANEKO SEEDS CO.,LTD, Maebashi, Gunma, Japan
5Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Hokkaido, Japan

Modern maize hybrids are commonly classified into fixed-ear or flex-ear types based on the plasticity of kernel number per ear (KNE) to environmental variation. This classification is potentially useful for achieving both high yield and stability across diverse field environments by enabling an adaptive response of sink size to resource availability and planting density. Understanding the morpho-physiological framework of ear plasticity is therefore critical for enhancing agronomic resilience of maize production under climate change. To this end, a multi-environment trial was conducted to characterize the response of sink traits in three fixed-ear and three flex-ear hybrids grown at five planting densities (5, 7, 9, 11, 13 plants m-2) across three locations in northern Japan. The representative fixed-ear and flex-ear hybrids were further compared for plant architecture in an on-station trial to identify key source traits potentially involved in ear plasticity and yield determination. In the MET, grain yield per plant at 5 plants m-2 in flex-ear hybrid was 43% greater than at 9 plants m-2, primarily due to increased KNE, whereas fixed-ear hybrids showed only a 29% increase. Among all the hybrids, one flex-ear hybrid exhibited the highest yield and yield stability. In the on-station trial, the structural analysis on this hybrid revealed a distinct leaf architecture, characterized by upper leaves with upright orientation and flatter, larger middle-leaves. This hybrid also exhibited significantly increased upper leaf area under low planting density, whereas the other hybrids did not display significant plasticity in leaf area among densities. Taken together, these findings suggest that flex-ear trait, combined with improved plant structure related to light interception, and morphological plasticity in leaves at upper canopy may play a crucial role in achieving high yield and its stability across planting densities.

P268: Unlocking sugar potential: Evaluating sugar content of waxy1/shrunken2-i hybrids

Quantitative Genetics & Breeding Danny Davis (Graduate Student)

Davis, Danielle T.1
Tracy, William F1

1Department of Plant and Agroecosystem Sciences, University of Wisconsin, Madison, WI 53706, USA

Waxy corn is a type of maize that creates an excess of amylopectin in the kernel due to the elimination of granule-bound starch synthase-I in the starch synthesis pathway​. This increase in amylopectin causes the kernel to have a gummy texture making it a popular eating corn in East Asia. The ears are harvested when at their milk stage and typically eaten after being steamed or processed into a dessert. Although waxy corn is eaten similarly to American sweet corn it isn’t sweet. Attempts have been made to rectify this by breeders. However, they result in cobs that segregate for kernel traits, leaving the consumer with a bite that has conflicting textures and flavor. By creating a double mutant stock with both wx1 and sh2-i we can develop a kernel that produces sucrose for a sweeter taste while maintaining a high amylopectin content for texture. This year, yield trials were grown with 4 hybrids with both the wx1 and sh2-i mutation. They were then harvested at the milk stage, 21 days after pollination. We measured the sugar content using high-throughput liquid chromatography. The check hybrid was homozygous recessive for wx1 dominant for Sh2. Ears from the same plot were also harvested at the mature stage and tested with iodine to see if there was an excess of amylopectin. Earlier experiments that measured the sugar content with a spectrometer showed that one of the hybrids had a high total sugar content of 30.3 percent of the kernel while maintaining a high amylopectin content. By increasing the sugar content in this popular eating corn we may make it more appealing to western markets and create a new niche for corn among consumers in East Asia.

P269: Vegetation indices including soil confound plant health and canopy cover: Multi-environment analysis of maize temporal progression

Quantitative Genetics & Breeding Fatma Ozair (Graduate Student)

Ozair, Fatma1
Murray, Seth1
DeSalvio, Aaron1
Arik, Mustafa1

1Texas A&M University, College Station, Texas, 77407

Vegetation indices (VIs) are valuable phenotypes for estimating crop health and for modeling crop growth temporally. Crop canopies capture sunlight promoting photosynthetic activity, thus contributing to overall biomass and grain yield potential. Plant breeders and agronomists utilize high throughput field data collected by unoccupied aerial vehicles (UAVs, also known as drones) to discover trends in complex traits, such as canopy cover and plant health. VI values are traditionally calculated without removing soil pixels, confounding the separate traits of plant health and canopy cover. The U.S. national maize (Zea mays L.) Genomes-to-Fields (G2F) dataset was used to evaluate canopy-adjusted VI models for three hybrid populations across 19 environments with 343 total flights. Heritability (repeatability) was used as an objective measure to compare VI estimates across different models. Results indicate that the modeling techniques are environment- and VI-dependent, with canopy-adjusted models outperforming traditional methods for specific subsets of the data. However, estimating these separately increased the repeatability by over 15% in some cases. Best linear unbiased predictors (BLUPs) for VIs are commonly used to track growth patterns, but they treat each flight date as discrete timepoints, limiting insights into growth trajectories and making it difficult to develop predictive scenarios. Canopy cover progresses directionally while plant health varies across time, but both differ across environments and have sparse and unaligned measurable timepoints. This requires techniques that can handle complex, limited temporal data. Functional principal component analysis (FPCA) was utilized as an alternative method to handle missing flight information across environments, and to enhance biological understanding of canopy and plant health. Understanding the patterns of canopy cover and plant health progression in maize will unlock insights on the optimal strategies for efficient accumulation of photosynthetic inputs/outputs under diverse environments.

P270: ZmNAGS enhances maize kernel size and grain yield through increasing photosynthesis and starch content

Quantitative Genetics & Breeding Huinan Li (Graduate Student)

Li, Huinan1
Luo, Yun1
Yang, Ning1
Yan, Jianbing1

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China

The kernel size of maize is a key trait that contributes greatly to grain yield. Cloning the genes for kernel size and dissecting the molecular mechanism of these genes will provide insights of the genetic basis and molecular regulation of kernel development, and provide target genes and theoretical basis for high-yield maize improvement. We used a recombinant inbred line (RIL) population derived from the cross between Zheng58 and SK for QTL mapping. By fine-mapped we cloned the candidate gene ZmNAGS, ZmNAGS is annotated as N-acetylglutamate synthase, which mainly acetylates glutamate to N-acetylglutamate and participates in the basal nitrogen metabolism of plants and promote plant growth. Through enzyme activity analysis and functional site identification, a single SNP variation (T/C) in the ZmNAGS coding region leads to an amino acid variation (Ser/Pro) and significantly affects protein acetylation ability. Therefore, T/C variation in the coding region is one of the functional sites of ZmNAGS affecting maize kernel size. The ZmNAGS protein is located in chloroplast and may regulate plant growth and development by affecting photosynthesis. Knocking out ZmNAGS resulted in a decrease in kernel length (KL), kernel width, and hundred kernel weight (HKW), as well as a reduction in maize ear size, plant height, leaf length and width, and a decrease in photosynthetic rate and chlorophyll content. Overexpression of ZmNAGS resulted in larger kernels, longer ears, increased plant height, as well as higher photosynthetic rate and chlorophyll content. In addition, RNA-seq analysis showed that differentially expressed genes were mainly enriched in the sugar transport pathway; metabolomic analysis showed that compared to the wild type, knocking out ZmNAGS had significantly reduced glucose, fructose, sucrose, 6-phosphate glucose content in the endosperm. At the same time, knocking out ZmNAGS, the starch content was significantly reduced. Proteome analysis showed that differentially abundant proteins in the overexpression lines were enriched in Calvin cycle and Glycolysis pathway. Futhermore, The KL, and grain yield of hybrid lines constructed with the overexpression line increased by 5.44%-7.63%, and 15.00%-20.11% respectively. In summary, we believe that ZmNAGS may regulate maize kernel size and yield by affecting photosynthesis and the accumulation of starch in the endosperm.

P271: qGSR2.02 confers recessive resistance to stalk rot and ear rot in maize

Quantitative Genetics & Breeding Chuan Chen (Graduate Student)

Chen, Chuan1 2 3
Zhong, Ling1 2 3
Zeng, Shirong1 2 3
Zhao, Daixin1 2 3
Zhang, Zhichao1 2 3
Yu, Yao1 2 3
Yang, Qin1 2 3

1College of Agronomy, Northwest A&F University; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100
2State Key Laboratory of Crop Stress Biology for Arid Areas; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100
3Key Laboratory of Maize Biology and Genetic Breeding in Arid Area of Northwest Region, Ministry of Agriculture; No. 3 Taicheng Road, Yangling, Shaanxi, China 712100

Gibberella stalk rot (GSR) and Gibberella ear rot (GER), both caused by fungal pathogen Fusarium graminearum, pose a devastating threat to global maize production. We have previously identified a major QTL for GSR resistance in maize bin 2.02 using a recombinant inbred line population derived from a cross between KA105 (resistant parent) and HZ4 (susceptible parent), designated as qGSR2.02. In this study, we validated phenotypic effect of qGSR2.02 in field trials using near-isogenic lines (NILs) developed from a heterogeneous inbred family. KA105-derived allele at qGSR2.02 significantly reduced GSR disease severity index (DSI) by 13–22% compared to lines with HZ4 allele. No significant difference was observed between the heterozygotes and homozygous HZ4 plants, suggesting that KA105 allele acts recessively at qGSR2.02. We also tested GER resistance using the two NILs, and found that NILKA105 showed significantly higher GER resistance than NILHZ4. Using a large segregating population, we fine-mapped qGSR2.02 to a 7-kb interval (KA105 genome) through recombinant-derived progeny testing strategy. Only one WD40 repeat gene, ZmWD40, is annotated in the qGSR2.02 region in susceptible parent HZ4. No corresponding gene is predicted in the 7-kb region in KA105 genome. Since the resistance allele is recessive, we hypothesis that ZmWD40 might be the causal gene at qGSR2.02. A maize mutant with a transposon insertion in the 5’-UTR region of the ZmWD40 gene showed significantly enhanced GSR resistance compared with its wildtype lacking the insertion. We are also making transgenic overexpression lines and knock-out lines to validate the function of ZmWD40. The findings will deepen our understanding of maize stalk rot and ear rot resistance and provide valuable resources for breeding program.