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

Location: Plant Genetics Research

2022 Annual Report

Objectives
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.

Approach
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
Objective 1. ARS researchers in Columbia, Missouri, finished last year’s milestones (for Sub-objective 1.1, the other milestones will not be completed due to vacancy). We are in the process of finishing the phenotyping and genome wide association studies (GWAS) now and will be well into writing the manuscript by the time this milestone is actually due (~9 months from the time of writing this). Objective 2. Over the past year, ARS researchers in Columbia, Missouri, have made significant progress on all Objective 2 Sub-objectives. For Sub-objective 2.1, ARS researchers in Columbia, Missouri, have planted maize mutants from the Chemistry Research Unit in Gainsville, Florida. For Sub-objective 2.2a, ARS researchers in Columbia, Missouri, have completed the evaluation of the northern corn rootworm wild strains on Bacillus thuringiensis (Bt) toxins Cry3Bb1 and Cry34/35 Bt. For Sub-objective 2.2b, ARS researchers have published that Cry34/35Ab1-selected colonies after removal from selection that resistance in western corn rootworm can disappear after the selection pressure is removed. For other traits, western corn rootworm has maintained resistance after selection pressure is removed. A series of studies have also begun in evaluating what to include in the next five-year Project Plan. Finally, for Sub-objective 2.3, ARS researchers have completed diet-toxicity assays we are now completing plant assays with wild northern corn rootworm. Objective 3. The scientist working on this objective left the agency; research in this objective has not been continued. Objective 4. Approximately 200 landrace/heirloom accessions had been chosen based on food properties described in the “Races of Maize” books and were sent to winter nurseries to create population hybrids, affectionately referred to as “corny combos” similar to the tomato “Heirloom Marriage” hybrids. This project complements the efforts outlined in Objective 4 by exploring the potential of landrace hybrids for unique kernel properties useful in food corn breeding. We finished the ear and kernel trait data collection from the 2020 trial, and we have harvested a final year of replicated trials from 2021 and are currently collecting the ear and kernel traits. The data are being analyzed and a manuscript is being prepared for submission. Objective 5. The genotyping in Sub-objective 5.1 was delayed by COVID closures, so milestones for this objective have been one year behind since 2020. We have now completed the genotyping, generated genetic maps for both populations, and have begun on the quantitative trait locus (QTL) analysis of highland adaptation traits. There are approximately 30 traits in three locations and for two populations, so our effort has focused on developing a pipeline for the most complex traits that can be simplified and automated for the complete dataset. Numerous QTL have been identified, several in common between populations and locations for macrohair traits related to highland adaptation. Two locations of Genomes to Fields Initiative (G2F) trials were planted in May 2022 (Sub-objective 5.2). These trials contain 542 and 461 unique hybrids, respectively, and are part of a larger experiment planted in partially replicated trials 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 which had been crossed with inbred lines with expired Plant Variety Protection certificates. The purpose of this year’s trials is to create a dataset for a novel diverse germplasm set that is the subject of the G2F Yield Prediction Contest. The field season is still underway and data collection is ongoing. Several models for predicting yield have been developed and published (Sub-objective 5.3). Another publication based on an improved model for maize will be submitted for publication before the end of the fiscal year. The wheat models have proven to be more difficult than originally anticipated due primarily to the lack of management data being recorded 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. Numerous crosses between distinct maize tetraploid varieties were made in the greenhouses (Sub-objective 5.4). Many of these crosses resulted in healthy seed which was planted in the field in May 2022 for phenotypic evaluation. We have completed the GWAS analysis on the maize primary data treated with and without synthetic auxin (Sub- objective 5.5). Primary root length was measured for mock treated and 2,4-D treated seedling roots. The absolute change, percent change, and ratio of root length between the mock treated and auxin treated roots were calculated. GWAS analysis was conducted on the five previously mentioned data measurements using publicly available software with 30,000 Single Nucleotide Polymorphisms (SNPs) and 1 million SNPs. Transcriptomic wide analysis was also done for the five data measurements with previously published transcriptomic data related to root gene expression. One significant SNP above the false positive threshold was identified on the short arm of chromosome 6 for the ratio of root length between mock treated and auxin treated primary root length. The other analyses did not identify significant SNPs associated with the phenotype, but there were some analyses that had SNPs that were close to the significance threshold. We have submitted Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) constructs for transformation into callus and should receive the T0 plantlets in August or September (Sub-objective 5.6). We are close to submitting additional CRISPR constructs for transformation.

Accomplishments
1. Rootworm resistance to Bacillus thuringiensis is linked to altered bacterial community. ARS and University of Missouri researchers in Columbia, Missouri, have evaluated the issue of corn rootworms that are rapidly developing resistance in the field to transgenic corn expressing rootworm-specific toxins. This threatens one of the few remaining management options that Corn Belt growers have for controlling this root chewing pest. The toxins kill corn rootworm larvae by damaging their intestinal lining, allowing microbes to leak from the intestine into the blood, causing a fatal infection. Therefore, the types of microbes present or missing within the rootworms could determine their sensitivity to poisons. In our study, we characterized the associated bacteria of toxin-resistant and toxin-susceptible western corn rootworm larvae. We found that resistant larvae harbored fewer bacteria and were distinct from those of susceptible larvae. Feeding on toxin-expressing maize by susceptible insects caused major changes in bacteria present, whereas the bacteria within resistant insects remained relatively unchanged. These results demonstrate that resistance to toxins is associated with bacterial alterations. Our work suggests that corn rootworm microbes could be manipulated by seed coatings, soil amendments or producer actions might limit development of resistance by this pest that causes $2 billion in losses by Corn Belt growers in terms of yield loss plus management costs.

Review Publications
Woore, M.S., Flint Garcia, S.A., Holland, J.B. 2021. The potential to breed a low-protein maize for protein-restricted diets. Crop Science. 61(6):4202-4217. https://doi.org/10.1002/csc2.20600.
Paddock, K.J., Pereira, A.E., Finke, D.L., Ericsson, A.C., Hibbard, B.E., Shelby, K. 2021. Host resistance to bacillus thuringiensis is linked to altered bacterial community within a specialist insect herbivore. Molecular Ecology. 30(21):5438-5453. https://doi.org/10.1111/mec.15875.
Shrestha, V., Yobi, A., Slaten, M.L., Chan, Y., Holden, S., Gyawali, A., Flint Garcia, S.A., Lipka, A.E., Angelovici, R. 2022. Multiomics approach reveals a role of translational machinery in shaping maize kernel amino acid composition. Plant Physiology. 188(1):111-133. https://doi.org/10.1093/plphys/kiab390.
Matthes, M.S., Darnell, Z., Best, N.B., Guthrie, K., Robil, J.M., Amstutz, J., Durbak, A., McSteen, P. 2022. Defects in meristem maintenance, cell division, and cytokinin signaling are early responses in the boron deficient maize mutant tassel-less1. Physiologia Plantarum. 174(2): Article e13670. https://doi.org/10.1111/ppl.13670.
Renk, J.S., Gilbert, A.M., Hattery, T.J., O'Connor, C.H., Monnahan, P.J., Anderson, N., Waters, A.J., Eickholt, D., Flint Garcia, S.A., Yandeau-Nelson, M.D., Hirsch, C.N. 2021. Genetic control of kernel compositional variation in a maize diversity panel. The Plant Genome. 14(3). Article e20115. https://doi.org/10.1002/tpg2.20115.
Weldekidan, T., Manching, H., Choquette, N., de Leon, N., Flint-Garcia, S.A., Holland, J.B., Lauter, N.C., Murray, S.C., Xu, W., Goodman, M., Wisser, R.J. 2022. Registration of tropical populations of maize selected in parallel for early flowering time across the United States. Journal of Plant Registrations. 16(1):100-108. https://doi.org/10.1002/plr2.20181.
Washburn, J.D., Strable, J., Dickinson, P., Kothapalli, S.S., Brose, J.M., Covshoff, S., Conant, G.C., Hibberd, J.M., Pires, C.J. 2021. Distinct C4 sub-types and C3 bundle sheath isolation in the Paniceae grasses. Plant Direct. 5(12): Article e373. https://doi.org/10.1002/pld3.373.
Washburn, J.D., Cimen, E., Ramstein, G., Reeves, T., O'Briant, P., McLean, G., Cooper, M., Hammer, G., Buckler IV, E.S. 2021. Predicting phenotypes from genetic, environment, management, and historical data using CNNs. Theoretical and Applied Genetics. 134:3997–4011. https://doi.org/10.1007/s00122-021-03943-7.
Volk, G.M., Byrne, P.F., Coyne, C.J., Flint Garcia, S.A., Reeves, P.A., Richards, C.M. 2021. Integrating genomic and phenomic approaches to support plant genetic resources conservation and use. Plants. 10(11). Article e2260. https://doi.org/10.3390/plants10112260.
Burns, M.J., Renk, J.S., Eickholt, D.P., Gilbert, A.M., Hattery, T.J., Holmes, M., Anderson, N., Waters, A.J., Kalambur, S., Flint Garcia, S.A., Yandeau-Nelson, M.D., Annor, G.A., Hirsch, C.N. 2021. Predicting moisture content during maize nixtamalization using machine learning with NIR spectroscopy. Theoretical and Applied Genetics. 134:3743–3757. https://doi.org/10.1007/s00122-021-03926-8.
Krishnan, H.B., Kim, S., Pereira, A.E., Jurkevich, A., Hibbard, B.E. 2022. Adenanthera pavonina, a potential plant-based protein resource: seed protein composition and immunohistochemical localization of trypsin inhibitors. Food Chemistry: X. 13. Article 100253. https://doi.org/10.1016/j.fochx.2022.100253.
Coates, B.S., Deleury, E., Gassmann, A.J., Hibbard, B.E., Meinke, L.J., Miller, N.J., Petzold-Maxwell, J., French, B.W., Sappington, T.W., Siegfried, B.D., Guillemaud, T. 2021. Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots. Biomed Central (BMC) Genomics. 22. Article 639. https://doi.org/10.1186/s12864-021-07932-4.
Huynh, M.P., Pereira, A.E., Geisert, R.W., Vella, M., Coudron, T.A., Shelby, K., Hibbard, B.E. 2021. Characterization of thermal and time exposure to improve artificial diet for western corn rootworm larvae. Insects. 12(9). Article 783. https://doi.org/10.3390/insects12090783.
Pereira, A.E., Huynh, M.P., Carlson, A.R., Haase, A., Kennedy, R.M., Shelby, K., Coudron, T.A., Hibbard, B.E. 2021. Assessing the single and combined toxicity of the bioinsecticide spear and cry3Bb1 protein against susceptible and resistant western corn rootworm larvae (coleoptera: chrysomelidae). Journal of Economic Entomology. 114(5):2220–2228. https://doi.org/10.1093/jee/toab160.
Perez-Limon, S., Li, M., Cintora-Martinez, G., Aguilar-Rangel, M., Salazar-Vidal, M., Gonzalez-Segovia, E., Blöcher-Juárez, K.A., Guerrero-Zavala, A., Barrales-Gamez, B., Carcaño-Macias, J.L., Costich, D.E., Nieto-Sotelo, J., Martinez-De La Vega, O., Simpson, J., Hufford, M.B., Ross-Ibarra, J., Flint Garcia, S.A., Diaz-Garcia, L., Rellán-Álvarez, R., Sawers, R.J. 2022. A B73 x palomero toluqueño mapping population reveals local adaptation in Mexican highland maize. G3, Genes/Genomes/Genetics. 12(3). Article jkab447. https://doi.org/10.1093/g3journal/jkab447.
Janzen, G.M., Aguilar-Rangel, M., Cintora-Martinez, C., Blöcher-Juárez, K., Gonzalez-Segovia, E., Studer, A.J., Runcie, D.E., Flint Garcia, S.A., Rellan-Alvarez, R., Sawers, R.J., Hufford, M.B. 2022. Demonstration of local adaptation of maize landraces by reciprocal transplantation. Evolutionary Applications. 15(5):817-837. https://doi.org/10.1111/eva.13372.
Paddock, K.J., Finke, D.L., Kim, K., Sappington, T.W., Hibbard, B.E. 2022. Patterns of microbiome composition vary across spatial scales in a specialist insect. Frontiers in Microbiology. 13. Article 898744. https://doi.org/10.3389/fmicb.2022.898744.
Tang, H.V., Berryman, D.L., Mendoza, J.S., Yactayo Chang, J.P., Li, Q., Christensen, S.A., Hunter III, C.T., Best, N.B., Soubeyrand, E., Akhtar, T., Basset, G.J., Block, A.K. 2022. Dedicated farnesyl diphosphate synthases circumvent isoprenoid-derived growth-defense tradeoffs in Zea mays. Plant Journal. https://doi.org/10.1111/tpj.15941.