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

Location: Plant Genetics Research

2020 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 at Columbia, Missouri, made substantial progress on re-tooling, repairing, troubleshooting, and testing the high-throughput root phenotyping robot, “Rootbot,” designed and created previously (Sub-objective 1.1). The phenotyping robot is nearly in working order and experiments aimed at understanding maize root growth genetics and physiology are expected to resume in autumn of 2020. Objective 2. Over the past year, ARS researchers at Columbia, Missouri, have made significant progress on all Objective 2 Sub-objectives. For Sub-objective 2.1, ARS researchers at Columbia, Missouri, have again planted the 282 maize inbred association panel (last year’s planting did not germinate due to heavy rain and cool temperatures after planting) and have analyzed data from the first year’s data where a series of genes have been identified that are associated with reduced rootworm damage in the field. For Sub-objective 2.2a, ARS researchers at Columbia, Missouri, have completed the evaluation of the northern corn rootworm laboratory colony on all current Bacillus thuringiensis (Bt) toxins currently targeting rootworm in plant assays and we have begun assays with wild Northern Corn Rootworm (NCR). For Sub-objective 2.2b, ARS researchers at Columbia, Missouri, have evaluated Cry34/35Ab1-selected colonies after removal from selection and documented for the first time 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. Reciprocal cross experiments have been initiated now that we have access to protein. Finally, for Sub-objective 2.3, ARS researchers in Columbia, Missouri, have completed diet assays for all current Bt toxins on the laboratory northern corn rootworm colony. The manuscript on baseline susceptibility of the northern corn rootworm to all Bt toxins in plant and diet assays has been sent to coauthors for review. We are now beginning diet-toxicity assays with wild NCR. Objective 4. ARS researchers at Columbia, Missouri, completed the 2019 evaluation of 80 landraces, resulting in a final dataset comprised of male/female flowering dates, plant/ear heights, leaf length/width, tassel morphology, tillering and ear number, ear morphology (husk color and number; ear diameter; number of kernel rows; cob length, width, and color), ear rot ratings, and kernel morphology (kernel color, width, and weight). This completes the early heirloom evaluations outlined in subobjective 4.1. Nineteen heirlooms/landraces were chosen from the landrace evaluation trial for population improvement in a collaboration between ARS researchers at Columbia, Missouri, and Raleigh, North Carolina. These populations are the starting materials for the breeding efforts in Sub-objective 4.1 and were planted in winter nursery to generate 40 families per landrace. These families were planted in June 2020, and selection will begin based on early seedling traits, followed by intermating and selection for adult plant traits and ear/kernel traits. Approximately 200 landrace/heirloom accessions were chosen based on food properties described in the “Races of Maize” books, ordered from the US National Plant Germplasm System, and were sent to a winter nursery for seed increase and crosses between landraces to form population hybrids, affectionately referred to as “corny combos.” 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. A replicated trial of 500 landraces and their landrace hybrids was planted in June 2020 in Columbia, Missouri, and data collection will begin shortly for agronomic, adaptation, and productivity traits. Objective 5. ARS researchers at Columbia, Missouri, have analyzed all the phenotypic trials for the highland by lowland landrace F2 populations from both Mexico and South America in Sub-objective 5.1 and the grant-funded, “The Genetics of Highland Adaptation in Maize” project (sub-ordinate project 5070-21000-041-06R). The leaf tissues have been prepared for genotypic analysis, and the design of the genotyping array is underway. The actual genotyping will be delayed by six to twelve months. A Genomes to Fields (G2F) trial of 1658 2-row yield plots was planted in June 2020, partially meeting the milestone for Sub-objective 5.2. The trial is comprised of three hybrid sets, with two replicates each. The hybrid sets represent stiff stalk doubled haploids crossed with three different non-stiff stalk testers planted at up to 30 locations (Missouri is only one location) to study how these hybrids respond to and interact with the environment. Stand counts appear to be poor in some parts of the trial due to extremely heavy rains after planting. At present, the hybrids have recently completed flowering and data have been collected on stand count, flowering time, and plant/ear height. Once plants reach full maturity, data will be collected on lodging and yield. Plant samples have been collected for a microbiome analysis coordinated by collaborators at the University of Georgia. An unmanned aerial vehicle (UAV) and an experimental field-based phenotyping rover were acquired and have been tested for taking high-throughput phenotyping data on the G2F trials. ARS personnel were licensed and trained in the use of this equipment. Image collection in the field is nearly complete and analysis of the images has begun. Computational resources and data were gathered, filtered, and analyzed to enable the development of Convolutional Neural Networks (CNNs) and Crop Growth Models (CGMs) for predicting cross-environment yields in maize (Sub-objective 5.3). Early stage models have been developed and are being tested and improved with the goal of predicting which maize cultivars perform best under different environmental conditions and why they perform that way. ARS researchers at Columbia, Missouri, collected publicly available high biomass maize lines from multiple public sources (Sub-Objective 5.4). Existing publicly available maize tetraploid lines (also known to display high biomass phenotypes) were collected. These high biomass lines were propagated and pollinated for increased seed numbers in the field and/or the greenhouse. Starting in autumn of 2020, the lines will be used to create and test new high biomass maize cultivars with potential utility as silage or bioenergy feedstocks.

Accomplishments
1. Western corn rootworm resistance is not fixed for all Bacillus thuringiensis (Bt) toxins. Western corn rootworms are a major insect pest of corn and are historically adept at evolving resistance to management practices, often without measurable fitness costs. Selection for resistance to all insecticides and most Bt toxins results in rapid development of resistance that remains fixed in rootworm populations. ARS and University of Missouri researchers in Columbia, Missouri, found that insect resistance to one of the major Bt toxins is associated with fitness costs including decreased lifespan and fecundity in adult insects. Recovery of susceptibility after removing toxin exposure for six generations to this one specific Bt toxin differs from rootworm resistance to all other toxins. This information is useful for devising strategies to prolong the duration of rootworm toxins in the field.

Review Publications
Seeve, C.M., Sunkar, R., Zheng, Y., Liu, L., Liu, Z., Mcmullen, M.D., Nelson, S.K., Sharp, R.E., Oliver, M.J. 2019. Water-deficit responsive microRNAs in the primary root growth zone of maize. Biomed Central (BMC) Plant Biology. 19:447. https://doi.org/10.1186/s12870-019-2037-y.
Gyawali, A., Shrestha, V., Guill, K.E., Flint Garcia, S.A., Beissinger, T.M. 2019. Single-plant GWAS coupled with bulk segregant analysis allows rapid identification and corroboration of plant-height candidate SNPs. Biomed Central (BMC) Plant Biology. 19:412. https://doi.org/10.1186/s12870-019-2000-y.
Huynh, M.P., Hibbard, B.E., Vella, M., Lapointe, S.L., Niedz, R.P., Shelby, K., Coudron, T.A. 2019. Development of an improved and accessible diet for western corn rootworm larvae using response surface modeling. Scientific Reports. 9:16009. https://doi.org/10.1038/s41598-019-52484-z.
Pereira, A.E., Ludwick, D.C., Barry, J.M., Meinke, L.J., Moellenbeck, D.J., Ellersieck, M.R., Reinders, J.D., Geisert, R.W., Hyte, K., Ernwall, A., Paddock, K.J., Hibbard, B.E. 2019. Optimizing egg recovery from wild northern corn rootworm beetles (coleoptera: chrysomelidae). Journal of Economic Entomology. 112(6):2737-2743. https://doi.org/10.1093/jee/toz234.
Zhang, X., Van Doan, C., Arce, C.C., Hu, L., Gruenig, S., Parisod, C., Hibbard, B.E., Herve, M.R., Nielson, C.N., Robert, C.A., Machado, R.A., Erb, M. 2019. Plant defense resistance in natural enemies of a specialist insect herbivore. Proceedings of the National Academy of Sciences. 116(46):23174-23181. https://doi.org/10.1073/pnas.1912599116.
Wisser, R.J., Fang, Z., Holland, J.B., Teixeira, J.E., Dougherty, J., Weldekidan, T., De Leon, N., Flint-Garcia, S.A., Lauter, N.C., Murray, S.C., Xu, W., Hallauer, A. 2019. The genomic basis for short-term evolution of environmental adaptation in maize. Genetics. 213(4):1479-1494. https://doi.org/10.1534/genetics.119.302780.
Ludwick, D.C., Ericsson, A.C., Meihls, L.M., Gregory, M.L., Finke, D.L., Coudron, T.A., Hibbard, B.E., Shelby, K. 2019. Survey of bacteria associated with western corn rootworm life stages reveals no difference between insects reared in different soils. Scientific Reports. 9:15332. https://doi.org/10.1038/s41598-019-51870-x.
Xue, W., Anderson, S.N., Wang, X., Yang, L., Crisp, P.A., Li, Q., Noshay, J., Albert, P.S., Birchler, J.A., Bilinski, P., Stitzer, M.C., Ross-Ibarra, J., Flint-Garcia, S.A., Chen, X., Springer, N.M., Doebley, J.F. 2019. Hybrid decay: A transgenerational epigenetic decline in vigor and viability triggered in backcross populations of teosinte with maize. Genetics. 213(1):143-160. https://doi.org/10.1534/genetics.119.302378.
Ramstein, G.P., Larsson, S.J., Cook, J.P., Edwards, J.W., Ersoz, E.S., Flint Garcia, S.A., Gardner, C.A., Holland, J.B., Lorenz, A.J., Mcmullen, M.D., Millard, M.J., Rocheford, T.R., Tuinstra, M.R., Bradbury, P., Buckler IV, E.S., Romay, M.C. 2020. Dominance effects and functional enrichments improve prediction of agronomic traits in hybrid maize. Genetics. 215:215-230. https://doi.org/10.1534/genetics.120.303025.
Falcon, C.M., Kaeppler, S.M., Spalding, E.P., Miller, N.D., Haase, N., Alkhalifah, N., Bohn, M., Buckler IV, E.S., Campbell, D.A., Ciampitti, I., Coffey, L., Edwards, J.W., Ertl, D., Flint Garcia, S.A., Gore, M.A., Graham, C., Hirsch, C.N., Holland, J.B., Jarquin, D., Knoll, J.E., Lauter, N.C., Lawrence-Dill, C.J., Lee, E.C., Lorenz, A., Lynch, J.P., Murray, S.C., Nelson, R., Romay, M., Rocheford, T., Schnable, P., Scully, B.T., Smith, M.C., Springer, N., Tuinstra, M., Walton, R., Weldekidan, T., Wisser, R.J., Xu, W., De Leon, N. Relative utility of agronomic, phenological, and morphological traits for assessing genotype-by-environment interaction in maize inbreds. Crop Science. 2020; 60:62-81. https://doi.org/10.1002/csc2.20035
Mcfarland, B.A., Alkhalifah, N., Bohn, M., Bubert, J., Buckler IV, E.S., Ciampitti, I., Edwards, J.W., Ertl, D., Gage, J.L., Falcon, C.M., Flint Garcia, S.A., Gore, M., Graham, C., Hirsch, C., Holland, J.B., Hood, E., Hooker, D., Jarquin, D., Kaeppler, S., Knoll, J.E., Kruger, G., Lauter, N.C., Lee, E.C., Lima, D.C., Lorenz, A., Lynch, J.P., Mckay, J., Miller, N.D., Moose, S.P., Murray, S.C., Nelson, R., Poudyal, C., Rocheford, T., Rodriguez, O., Romay, M., Schnable, J.C., Schnable, P.S., Scully, B.T., Sekhon, R., Silverstein, K., Singh, M., Smith, M., Spalding, E.P., Springer, N., Thelen, K., Thomison, P., Tuinstra, M., Wallace, J., Walls, R., Wills, D., Wisser, R.J., Xu, W., Yeh, C., De Leon, N. Maize genomes to fields (G2F): 2014 –2017 field seasons: genotype, phenotype, climatic, soil and inbred ear image datasets. BMC Research Notes. 13,71 (2020). https://doi.org/10.1186/s13104-020-4922-8.
Pereira, A.E., Huynh, M.P., Sethi, A., Miles, A.L., French, B.W., Ellersieck, M.R., Coudron, T.A., Shelby, K., Hibbard, B.E. 2020. Baseline susceptibility of a laboratory strain of northern corn rootworm, diabrotica barberi (coleoptera: chrysomelidae) to bacillus thuringiensis traits in seedling, single plant, and diet-toxicity assays. Journal of Economic Entomology. 113(4):1955–1962. https://doi.org/10.1093/jee/toaa107.