Skip to main content
ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Research Project #434241

Research Project: Genetic and Physiological Mechanisms Underlying Complex Agronomic Traits in Grain Crops

Location: Plant Genetics Research

2023 Annual Report

Objective 1: Identify genetic and physiological mechanisms controlling growth under drought in maize, wheat, and related species. • Sub-objective 1.1: Characterize the genetic regulation of maize root growth responses to soil water-deficit stress. • Sub-objective 1.2: Determine the roles of plant hormones abscisic acid (ABA) and gibberellins (GA) in the regulation of wheat root responses to water deficit. • Sub-objective 1.3: Characterize the genetic networks that link transcription factor expression and metabolism central to cellular protection during dehydration in a C4 resurrection grass. Objective 2: Characterize corn for natural rootworm resistance, rootworm larvae for Bt tolerance, and artificial diets for improved understanding of rootworm biology and management. • Sub-objective 2.1: Systematically screen exotic and Germplasm Enhancement of Maize (GEM) germplasm, identify potential sources of western corn rootworm (WCR) resistance, verify resistance, and move into adapted germplasm. • Sub-objective 2.2: Characterize heritability and other traits of rootworm larvae with Bt tolerance. • Sub-objective 2.3: Evaluate northern corn rootworm (NCR) development on larval Diabrotica diets and develop a diet toxicity assay for NCR. Objective 3: Identify genetic and physiological mechanisms governing response to artificial selection in cereals and related species. • Sub-objective 3.1: Develop an experimental evolution maize population to characterize adaptation to selective pressures at the genomic level in maize and related species. • Sub-objective 3.2: Quantify the importance of epistasis with novel Epistasis Mapping Populations. • Sub-objective 3.3: Develop, implement, and validate statistical methods to better understand traits controlled by multiple genes acting in concert. Objective 4: Develop and characterize germplasm to elucidate the genetic mechanisms underlying nutritional and food traits in maize. • Sub-objective 4.1: Screen and develop maize germplasm for traits important in food-grade corn. Objective 5: Identify genetic and physiological mechanisms underlying maize adaptation to the environment to enhance its productivity. • Sub-objective 5.1: Develop and evaluate germplasm segregating for adaptation to high elevation. • Sub-objective 5.2: Evaluate diverse maize hybrids in multi-location trials as part of the Genomes To Fields Genotype x Environment Project.

Conduct genome-wide association analysis of water-stress root growth using high-throughput maize root phenotyping to link transcription factor (TF) expression with root growth phenotypes under stress. Characterize water deficit growth and hormone responses in wheat roots, and interrogate the gene expression profiles (RNAseq) for the root growth zone. Use chromatin immunoprecipitation-sequencing to establish the role of transcription and TF targets in the response of both wheat and maize roots to water deficits. Develop gene network maps for dehydration TFs in the resurrection grass Sporobolus stapfianus. Evaluate 75 new sources of maize germplasm each year for resistance to Western Corn Rootworm (WCR) larval feeding in replicated field trials. Develop an artificial diet for Northern Corn Rootworm (NCR) and conduct toxicity assays for all available Bt proteins. Expose NCR populations to current industry Bt corn in plant assays and measure the effect on insect development. Evaluate the inheritance of Bt resistance in WCR. Conduct five cycles of selection for high and low plant height in the Shoepeg maize landrace population, followed by genotyping and selection mapping. Phenotype an Epistasis Mapping Population and conduct statistical tests for epistatic effects. Screen 100 heirloom maize varieties for adaptation to the southern Corn Belt and make selections based on agronomic performance and kernel composition traits. Create and release modified open pollinated varieties with improved performance and food characteristics. Conduct quantitative trait locus (QTL) mapping of traits related to highland adaptation in maize populations grown at low, mid, and high elevations. Compare QTLs identified in a Mexican and South American germplasm. Identify candidate genes based on traits related to adaptation and fitness at varying elevation. Participate in multi-location yield trials to evaluate diverse maize hybrids across the US.

Progress Report
This is the final report for this project which terminated in February 2023. Research will be continued under the new project 5070-21220-046-000D, “Adaptation of Grain Crops to Varying Environments Including Climates, Stressors, and Human Uses”. The project had numerous changes over the five year cycle: departure of a scientist in 2018, retirement of a scientist in 2019, and the hiring of two scientists in 2020 and 2021. Despite these changes in personnel, the previous five years resulted in 80 publications. Objective 1 Over the life of the project, Sub-objective 1.1 was impacted by several delays including the retirement of the scientist in charge of the sub-objective in 2019, the re-assignment of the sub-objective to a newly hired scientist in January 2020, and significant delays due to shutdown of in-person laboratory experiments at the beginning of the COVID-19 pandemic. However, the project still resulted in completion of the “Rootbot,” a high-throughput root phenotyping robot, establishment of a protocol for reliable and reproducible measurements of root growth patterns, screening of the maize 282 inbred association panel, one published manuscript, one accepted manuscripts awaiting publication, and one manuscript in progress for likely submission in FY2024. Sub-objective 1.2 had limited results as there was a critical vacancy followed by retirement of the scientist. Sub-objective 1.3 resulted in the construction of expression vectors for the root active and water-deficit responsive transcription factors, reannotation of the resurrection grass genome, and identification of transcription factor sequences. Subordinate project 5070-21000-038-09R resulted in a completed global metabolite profile for regions of the nodal root growth zone. Retirement of the scientist limited the results of this sub-objective and its subordinate project. Objective 2 All of the research in this objective has been completed or is substantially complete. We have been diligently moving forward and making good progress on the new proposed project plan since then. During the life of the project, Sub-objective 2.1 resulted in the evaluation of western corn rootworm (WCR) damage in the 282 inbred association panel, 100 maize lines from the Plant Introduction Station, and maize mutants from the ARS location in Gainesville, Florida. Sub-objective 2.2 resulted in evaluation of the northern corn rootworm (NCR) laboratory colony and wild NCR populations on Bacillus thuringiensis (Bt) toxins Cry3Bb1 and Cry34/35, and documentation that resistance to Cry34/35Ab1 in WCR can disappear after the selection pressure is removed. Four publications resulted directly related to this sub-objective, and numerous others. Sub-objective 2.3 resulted in diet-toxicity assays for all current Bt toxins on both the laboratory colony and wild populations of NCR. A manuscript on baseline susceptibility of the NCR to all Bt toxins was published. Objective 3 The scientist working on this objective left the agency within a month of project certification; the research was abandoned. Objective 4 Sub-objective 4.1. Over the past year, kernel protein and starch contents have been estimated using near infra-red (NIR) reflectance for the 2019, 2020, and 2021 grow-outs of the “Heirloom Marriage” experiment. We are moving forward on the new project plan; seed increases of the U.S. landraces is underway in summer 2023. During the life of the project, Sub-objective 4.1 resulted in evaluation several sets of maize landraces (heirlooms) for many plant, ear, and kernel traits including 60 temperate landraces (one published manuscript) and the Heirloom Marriage Experiment comprised of 130 Mexican and South American landraces and nearly 500 landrace hybrids (analysis underway). Twelve heirlooms were subjected to two cycles of selection for improved agronomics; the original heirlooms and the improved populations were evaluated in order to measure the response to selection for the target traits and to verify that the desirable traits in each population have not changed. Yield trials are currently underway for the twelve heirlooms. In addition, germplasm was screened for low protein and low phenylalanine in order to identify varieties to broaden food options for people with phenylketonuria, resulting in a manuscript. Objective 5 Sub-objective 5.1. In FY2023, analysis of the highland adaptation populations continued. Sub-objective 5.2. Two locations of the Genomes to Fields Initiative (G2F) trials were planted in 2022, but one location failed and was abandoned due to weather and the resulting poor germination. The other location was successfully grown and phenotyped. This trial contained 461 unique hybrids and was part of a larger experiment planted at over 25 locations across the United States. Most of the hybrids are LH244 testcrosses of doubled haploids derived from four Germplasm Enhancement of Maize releases crossed with inbred lines with expired Plant Variety Protection certificates. The purpose of 2022 trials was to create a dataset for a novel diverse germplasm set for the G2F Yield Prediction Contest. A world-wide phenotyping competition was held by the G2F initiative at the end of 2022. ARS researchers in Columbia, Missouri, lead the organization of the competition, dataset curation, and other aspects of the competition. Two locations of the 2023 G2F trials were planted in Columbia, Missouri, in May; both locations germinated well and data collection is ongoing. Also related to Sub-objective 5.2 (and Objective 1 in the new project plan) is the sub-ordinate project “CERCA - Circular Economy that Reimagines Corn Agriculture.” We planted field trials for examining nitrogen (N) and protein content in the grain in order to identify low-N germplasm for subsequent experiments to reduce the negative environmental impacts of corn agriculture. We also planted experiments to examine source (plant) and sink (ear/kernel) relationships for nitrogen. Sub-objective 5.3. Several models for predicting yield were developed and published based on work from the final year of the five-year project. Sub-objective 5.4. Numerous crosses between distinct maize tetraploid varieties were made in the greenhouses and planted in the field for evaluation. Sub-objectives 5.5 and 5.6. We received T0 plantlets for the gene edited mutant lines DWARF9, BZR1, BZR10, BZR7, and BZR11. These lines are being outcrossed to several inbred backgrounds. We are currently genotyping the edited lines to identify the genetic modifications and characterize the effect on plant growth and development. We have planted B73 seeds in the field to perform our first round of pollen mutagenesis to develop stocks of highly mutagenized lines to be sequenced in year 4 of the upcoming project plan. We are developing protocols to quantify endogenous hormone levels in maize tissue. We have fully optimized a brassinosteroid quantification protocol that includes a derivatization step with picolinic acid. In addition to being able to measure downstream brassinosteroids, we are also able to quantify upstream structural sterols. We are in the process of developing a single extraction protocol to measure cytokinins, auxin, abscisic acid, salicylic acid, strigolactones, gibberellins, jasmonic acid, and benzoxazinoids using liquid chromatography and mass spectrometry. During the life of the project, Sub-objective 5.1 resulted in the creation of two highland by lowland landrace F2 populations and evaluation of these populations in low-, mid-, and high-elevation field sites for adaptation traits. The populations were genotyped, and genetic maps were constructed followed by preliminary quantitative trait locus (QTL) analysis. Analysis was delayed by the pandemic, but analysis is ongoing. Over 100 hybrids of landrace crossed with B73 were created and evaluated in the same low- and high-elevation field sites, and allele specific gene expression was used to identify genes associated with highland adaptation. Three publications have come from this sub-objective to date. Sub-objective 5.2 efforts focused on evaluating thousands of hybrid plots as part of G2F: 1600 plots in 2018, 800 plots in 2019, 1594 plots in 2020, 550 plots in 2021, and 600 plots in 2022, and the 2023 field season is underway. An unmanned aerial vehicle (UAV) and an experimental field-based phenotyping rover were acquired in 2020, and protocols and pipelines were developed for storing, sharing, analyzing, and archiving high throughput phenotyping data. This resulted in five manuscripts published over this five-year period. Sub-objective 5.3 resulted in Convolutional Neural Networks (CNNs) and Crop Growth Models (CGMs) for predicting cross-environment yields in maize and wheat. Several models for predicting maize yield have been developed and three manuscripts have been published to date. The wheat models have proven to be more difficult due to the lack of management data in the wheat breeding databases. If better data sources can be found and refined, then the models from maize will be applied to these wheat data. Sub-objective 5.4 focused on acquiring and propagating publicly available high biomass maize lines from multiple public sources. New maize tetraploid lines were created from diploid inbred lines, and numerous crosses between distinct maize tetraploid varieties were made in the greenhouses. Many of these crosses resulted in healthy seed that was planted in the field for phenotypic evaluation. Sub-objective 5.5 developed phenotyping protocols to screen and phenotype maize roots for sensitivity to auxin treatment. Seed of the 282 inbred line association panel was treated with and without synthetic auxin, the roots imaged, and primary root lengths were extracted. Genome wise association analysis was conducted, and transcriptome wide analysis was also done using previously published transcriptomic data related to root gene expression. Three manuscripts were published.

1. World-wide competition resulted in the development of improved publicly available yield prediction methods and models. Predicting how a new crop cultivar will yield in a new location is critical for both plant breeders who develop new cultivars and the farmers who grow them. Even small improvements in yield prediction have the potential to save millions of dollars and make farming systems more efficient and sustainable. An ARS scientist in Columbia, Missouri, played a leading role in developing and curating the largest public maize dataset of its kind. They also organized a global maize yield prediction competition challenging the global community to develop and test new yield prediction methods. The competition was run by the Genomes to Field initiative, the National Corn Growers Association, and Iowa Corn ( with support from additional ARS scientists. Dozens of new yield prediction strategies and models were developed for public release. Some of these models are more accurate than the current state of the art, while others provide examples of theoretically compelling strategies that did not perform well in practice, critical knowledge in a field where the number of promising modeling strategies to be tested and eliminated is enormous. The developed models and datasets provide improved strategies for modeling and predicting yield with the potential to improve agricultural productivity and efficiency.

2. Gibberellin control of plant height and reproductive development can be uncoupled to utilize these plant growth regulators to improve maize architecture. The green revolution reduced plant height to increase yields, primarily in wheat and rice. The reduced height trait occurred by reducing gibberellin response. This trait that results from reducing gibberellin response in maize has additional deleterious effects on reproductive development where the male flowers are retained and the persistence of these male flowers in the female ear increases the humidity in the husk leaves and can cause disease problems. Therefore, the reduced height trait has not been utilized in maize. ARS scientists in Columbia, Missouri, determined that gibberellin regulation of plant height and reproductive development does not act through the same pathways, offering the possibility that gibberellin effects on plant height and reproduction can be separated. This suggests that the positive effect on reducing plant height can be achieved in maize using these green revolution genes from wheat and rice. Targeting and modifying the genetic factors responsible for the persistence of male flowers is a valuable method to achieve this feat. These findings provide a pathway to improve maize architecture and alleviate some yield losses from severe weather caused by climate change.

Review Publications
Jabeur, R., Guyon, V., Toth, S., Pereira, A.E., Huynh, M.P., Selmani, Z., Boland, E., Bosio, M., Beuf, L., Clark, P., Vallenet, D., Achouak, W., Audiffrin, C., Torney, F., Paul, W., Heulin, T., Hibbard, B.E., Toepfer, S., Sallaud, C. 2023. A novel binary pesticidal protein from Chryseobacterium arthrosphaerae controls western corn rootworm by a different mode of action to existing commercial pesticidal proteins. PLOS ONE. 18(2). Article e0267220.
Best, N.B., Dilkes, B.P. 2022. Transcriptional responses to gibberellin in the maize tassel and control by DELLA domain proteins. The Plant Journal. 112(2):493-517.
Hu, H., Crow, T., Nojoomi, S., Schulz, A.J., Estevez-Palmas, J.M., Hufford, M., Flint Garcia, S.A., Sawers, R., Rellan-Alvarez, R., Ross-Ibarra, J., Runice, D. 2022. Allele-specific expression reveals multiple paths to highland adaptation in maize. Molecular Biology and Evolution. 39(11). Article msac239.
Best, N.B., McSteen, P. 2022. Mapping maize mutants using bulked-segregant analysis and next-generation sequencing. Current Protocols in Plant Biology. 2(11). Article e591.
Pereira, A.E., Geisert, R.W., Hibbard, B.E. 2022. Maize inbred Mp708 is highly susceptible to western corn rootworm, diabrotica virgifera virgifera (coleoptera: chrysomelidae), in field and greenhouse assays. Journal of Insect Science. 22(6). Article 8.
Paddock, K.J., Dellamano, K., Hibbard, B.E., Shelby, K. 2023. eCry3.1Ab-resistant western corn rootworm larval midgut epithelia respond minimally to Bt intoxication. Journal of Economic Entomology. 116(1):263-267.
Pereira, A.E., Huynh, M.P., Paddock, K.J., Ramirez, J.L., Caragata, E.P., Dimopoulos, G., Krishnan, H.B., Schneider, S.K., Shelby, K., Hibbard, B.E. 2022. Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis. Scientific Reports. 12. Article 17858.
Boateng, I.D., Kuehnel, L., Daubert, C.R., Agliata, J., Zhang, W., Kumar, R., Flint Garcia, S.A., Azlin, M., Somavat, P., Wan, C. 2023. Updating the status quo on the extraction of bioactive compounds in agro-products using a two-pot multivariate design. A comprehensive review. Food and Function. 2(14):569-601.
Huynh, M.P., Hibbard, B.E., Ho, K., Shelby, K. 2022. Toxicometabolomic profiling of resistant and susceptible western corn rootworm larvae feeding on Bt maize seedlings. Scientific Reports. 12. Article 11639.
Kick, D.R., Wallace, J.G., Schnable, J.C., Kolkmann, J.M., Alaca, B., Beissinger, T.M., Edwards, J.W., Ertl, D., Flint-Garcia, S.A., Gage, J.L., Hirsch, C.N., Knoll, J.E., de Leon, N., Lima, D.C., Moreta, D., Singh, M.P., Thompson, A., Weldekidan, T., Washburn, J.D. 2023. Yield prediction through integration of genetic, environment, and management data through deep learning. G3, Genes/Genomes/Genetics. 13(4). Article jkad006.
Geisert, R.W., Huynh, M.P., Pereira, A.E., Shapiro Ilan, D.I., Hibbard, B.E. 2023. An improved bioassay for the testing of entomopathogenic nematode virulence to the western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae): with focus on neonate insect assessments. Journal of Economic Entomology. 116(3): 726-732.
Lima, D., Castro Aviles, A., Alpers, T., Mcfarlan, B., Kaeppler, S., Ertl, D., Romay, C., Gage, J., Holland, J.B., Beissinger, T., Bohn, M., Buckler, E., Edwards, J., Flint-Garcia, S., Hirsch, C., Hood, E., Hooker, D., Knoll, J., Kolkman, J., Liu, S., Mckay, J., Minyo, R., Moreta, D.E., Murray, S., Nelson, R., Schnable, J., Sekhon, R., Singh, M., Thomison, P., Thompson, A., Tuinstra, M., Wallace, J., Washburn, J.D., Weldekidan, T., Wisser, R., Xu, W. 2023. 2018-2019 field seasons of the maize genomes to fields (G2F) G x E project. BMC Genomic Data. 24:29.
Lima, D.C., Washburn, J.D., Varela, J.I., Chen, Q., Gage, J.L., Romay, M.C., Holland, J.B., Ertl, D., Lopez-Cruz, M., Aguate, F.M., De Los Campos, G., Kaeppler, S., Beissinger, T., Bohn, M., Buckler IV, E.S., Edwards, J.W., Flint Garcia, S.A., Gore, M.A., Hirsch, C.N., Knoll, J.E., Mckay, J., Minyo, R., Murray, S.C., Ortez, O.A., Schnable, J., Sekhon, R.S., Singh, M.P., Sparks, E.E., Thompson, A., Tuinstra, M., Wallace, J., Weldekidan, T., Xu, W., De Leon, N. 2023. Genomes to fields 2022 maize genotype by environment prediction competition. BMC Research Notes. 16: Article 148.
Chan, Y., Dietz, N., Zeng, S., Wang, J., Flint Garcia, S.A., Salazar-Vidal, N.M., Skrabisova, M., Bilyeu, K.D., Joshi, T. 2023. The allele catalog tool: a web-based interactive tool for allele discovery and analysis. BMC Genomics. 24: Article 107.
Guan, J., Li, C., Flint Garcia, S.A., Suzuki, M., Wu, S., Saunders, J., Dong, L., Bouwmeester, H., Mccarty, D., Koch, K. 2023. Maize domestication phenotypes reveal strigolactone networks coordinating grain size evolution with kernel-bearing cupule architecture. The Plant Cell. 35(3):1013-1037.
Pittas, A.G., Kawahara, T., Jorde, R., Dawson-Hughes, B., Vickery, E.M., Angellotti, E., Nelson, J., Trikalinos, T.A., Balk, E.M. 2023. Vitamin D and risk for type 2 diabetes in people with prediabetes: a systematic review and meta-analysis of individual participant data from 3 randomized clinical trials. Annals of Internal Medicine.
Boateng, I.D., Kumar, R., Daubert, C.R., Flint Garcia, S.A., Mustapha, A., Kuehnel, L., Agliata, J., Li, Q., Wan, C., Somavat, P. 2023. Sonoprocessing improves phenolics profile, antioxidant capacity, structure, and product qualities of purple corn pericarp extract. Ultrasonics Sonochemistry. 95: Article 106418.
Flint Garcia, S.A., Feldmann, M., Dempewolf, H., Morrell, P.L., Ross-Ibarra, J. 2023. Diamonds in the not-so-rough: wild relative diversity hidden in crop genomes. PLoS Biology. 21(7): Article e3002235.
Best, N.B., Dilkes, B. 2023. Genetic evidence that brassinosteroids suppress pistils in the maize tassel independent of the Jasmonic acid pathway. Plant Direct. 7(7): Article e501.