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

Location: Plant Genetics Research

2019 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. With the completion of the “Rootbot,” the high-throughput root phenotyping robot, ARS researchers in Columbia, Missouri, have established a protocol for reliable and reproducible measurements of root growth patterns under well-watered and water-deficit conditions (sub-objective 1.1). ARS researchers in Columbia, Missouri, are currently working through the 282 maize lines that constitute the Maize 282 association panel and have completed a substantial number. The full panel is expected to be complete by summer 2019. ARS researchers in Columbia, Missouri, have almost completed the construction of the expression vectors for the root active and water-deficit responsive transcription factors using a vector that adds an antibody recognition site for in vitro expression and protein production. The expressed proteins will be used in a deoxyribonucleic acid affinity purification sequencing assay, a technique that identifies transcription factor binding sites on deoxyribonucleic acid, to determine what genes each maize transcription factor binds to and controls within the response of maize roots to a water deficit. The unit has a critical vacancy and so there has been no progress on Sub-objective 1.2. ARS researchers in Columbia, Missouri, have completed a reannotation of the resurrection grass genome and have identified the correct transcription factor sequences they have targeted for deoxyribonucleic acid affinity purification sequencing analysis (sub-objective 1.3). ARS researchers in Columbia, Missouri, are continuing the cloning of 18 root transcription factors and once cloned they will be placed in appropriate vectors for the deoxyribonucleic acid affinity purification sequencing assays. ARS researchers in Columbia, Missouri, have also undertaken a network analysis of the co-expressed genes related to the ability of the resurrection grass to survive drying. This network analysis is based on transcription factors as hubs in the network and this will add to the ability to map what genes are involved in the genetic control of dehydration tolerance. ARS researchers in Columbia, Missouri, have completed the global metabolite profiling for regions of the nodal root growth zone from our targeted maize line (subordinate project 5070-21000-038-09R). The data has been compiled and initial analyses performed and ARS researchers in Columbia, Missouri, are in the process of assessing the metabolomes for hypothesis generation. 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 again planted the 282 maize inbred association panel 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 in Columbia, Missouri, have completed the evaluation of the northern corn rootworm laboratory colony on all current Bt toxins currently targeting rootworm in plant assays. For Sub-objective 2.2b, ARS researchers in 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. 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. Objective 4. The 2018 evaluation of 60 landraces was completed, thus completing the 12-month milestones in Objective 4. The final dataset was 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 data was used to choose the landraces that would be targeted for the breeding project to produce improved landraces. A second year of the landrace evaluation experiment (24-month milestone of Objective 4) was planted in late May 2019: three replicates of 80 landraces were planted in both Missouri and North Carolina. Pollinations are underway in order to produce grain for compositional analysis, as well as data collection for an array of adaptation and agronomic traits as described above. After harvest, an array of ear and kernel phenotypes will also be collected like the 2018 trial. Approximately 130 landrace accessions were chosen based on food properties described in the “Races of Maize” books, ordered from the U.S. National Plant Germplasm System, and were sent to a winter nursery for seed increase and crosses between landrace 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 in food corn breeding. The government shutdown interfered with the timing of travel to the winter nursery and making crosses among the landraces, resulting in only 60 population hybrids being produced. More hybrids will be attempted in summer 2019 and winter 2019-2020. The 60 population hybrids and the original 130 landraces were planted in a replicated trial in Columbia, Missouri, and are being evaluated for agronomic, adaptation, and productivity traits. Objective 5. In collaboration with researchers at the National Laboratory of Genomics for Biodiversity (Langebio) in Irapuato, Mexico, and part of the grant-funded “The Genetics of Highland Adaptation in Maize” project (subordinate project 5070-21000-041-06R), all phenotypic trials have been completed for the highland by lowland landrace F2 populations from both Mexico and South America, thus completing the 24 month milestone of Sub-objective 5.1 well ahead of schedule. Both populations were planted in the mid-elevation site (Ameca, Jalisco) and the high-elevation site (Metepec, Mexico) in 2018, with two replicates at each location. An ARS researcher and a technician in Columbia, Missouri, traveled to both sites along with other grant project personnel to collect plant phenotypes (male and female maturity, plant and ear height, tassel length and number tassel branches). Ear and kernel data collection (number of ears, total kernel mass per plant, and fifty kernel mass) were collected on the harvested ears. Phenotypic data analysis is currently underway. A Genomes to Fields (G2F) trial of 800 2-row yield plots was planted in late May 2019, meeting the milestone for Sub-objective 5.2. The trial is comprised of three hybrid sets, with two replicates each. The hybrid sets represent populations of “good, bad, and ugly” hybrids planted at nearly 30 locations (Missouri is only one location) to study how these hybrids respond to and interact with the environment. Stand counts were somewhat poor due to extremely heavy rains after planting. Plant samples were collected for a microbiome analysis coordinated by collaborators at the University of Georgia. At present, only the earliest of the hybrids have flowered, but data will be collected for maturity, plant/ear height, lodging and yield as the season progresses.

Accomplishments
1. Susceptibility restored for Bt-resistant rootworm. The western corn rootworm has evolved resistance to nearly all management tactics targeting it, including Bt corn. Using rootworm strains resistant and susceptible to Bt, ARS researchers in Columbia, Missouri, evaluated ribonucleic acid produced after each were fed Bt corn or non-Bt corn. Several target genes that were differently expressed between resistant and susceptible rootworm strains were evaluated further by producing double stranded ribonucleic acid to knockdown the target genes. Transcripts of all four genes were significantly knocked down in both rootworm strains. Finally, resistant and susceptible larvae were exposed to Bt toxin in presence or absence of double stranded ribonucleic acid. The double stranded ribonucleic acid of one of the genes restored significant susceptibility to the resistant rootworm strain while susceptible rootworms maintained full susceptibility after exposure. This is the first-time susceptibility to Bt has been restored after knocking down a resistance gene and could be applied by expressing the double stranded ribonucleic acid in corn along with Bt or by better understanding resistance for future management tools.

Review Publications
Zhao, Z., Miehls, L.M., Hibbard, B.E., Ji, T., Elsik, C., Shelby, K. 2019. Differential gene expression in response to eCry3.1Ab ingestion in an unselected and eCry3.1Ab-selected western corn rootworm (Diabrotica virgifera virgifera LeConte) population. Scientific Reports. 9:4896. https://doi.org/10.1038/s41598-019-41067-7.
Man, H.P., Hibbard, B.E., Lapointe, S.L., Niedz, R.P., French, B.W., Pereira, A.E., Finke, D.L., Shelby, K., Coudron, T.A. 2019. Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae. Scientific Reports. 9:3709. https://doi.org/10.1038/s41598-019-39709-x.
Pereira, A.E., Coudron, T.A., Shelby, K., French, B.W., Bernkalu, E.J., Bjostad, L.B., Hibbard, B.E. 2019. Comparative susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) neonates to selected insecticides and Bt proteins in the presence and absence of feeding stimulants. Journal of Economic Entomology. 112(2):842-851. https://doi.org/10.1093/jee/toy415.
Guo, T., Yu, X., Li, X., Zhang, H., Zhu, C., Flint-Garcia, S.A., McMullen, M.D., Holland, J.B., Szalma, S.J., Wisser, R., Yu, J. 2019. Optimal designs for genomic selection in hybrid crops. Molecular Plant. 12(3):390-401. https://doi.org/10.1016/j.molp.2018.12.022.
Mutiga, S.K., Chepkwony, N., Hoekenga, O.A., Flint-Garcia, S.A., Nelson, R.J. 2019. The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus. Plant Breeding. 138(1):38-50. https://doi.org/10.1111/pbr.12672.
Glowinski, A., Flint-Garcia, S.A. 2018. Germplasm resources for mapping quantitative traits in maize. In: Bennetzen, J., Flint-Garcia, S., Hirsch, C., Tuberosa, R.(eds). The Maize Genome. Springer International Publishing AG. Cham, Switzerland. p.143-159.
Geisert, R.W., Ludwick, D.C., Hibbard, B.E. 2019. Effects of cold storage on nondiapausing eggs of the western corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology. 112(2):708-711. https://doi.org/10.1093/jee/toy405.
Guyer, A., Hibbard, B.E., Holzkamper, A., Erb, M., Robert, C.A. 2018. Influence of drought on plant performance through changes in belowground tritrophic interactions. Ecology and Evolution. 8:6756-6765. http://doi.org/10.1002/ece3.4183.
Hiltpold, I., Hibbard, B.E. 2018. Indirect root defenses cause induced fitness costs in Bt-resistant western corn rootworm. Journal of Economic Entomology. 111(5):2349-2358. https://doi.org/10.1093/jee/toy220.
Bernklau, E.J., Hibbard, B.E., Bjostad, L.B. 2018. Sugar preferences of western corn rootworm larvae in a feeding stimulant blend. Journal of Applied Entomology. 142(10):947-958. https://doi.org/10.1111/jen.12540.
Meihls, L., Huynh, M.P., Ludwick, D.C., Coudron, T.A., French, B.W., Shelby, K., Hitchon, A.J., Smith, J.L., Schaafsma, A.W., Pereira, A.E., Hibbard, B.E. 2018. Comparison of six artificial diets for western corn rootworm bioassays and rearing. Journal of Economic Entomology. 111(6):2727-2733. https://doi.org/10.1093/jee/toy268.
Jaffuel, G., Imperiali, N., Shelby, K., Campos-Herrera, R., Geisert, R.W., Maurhofer, M., Loper, J.E., Keel, C., Turlings, T.C., Hibbard, B.E. 2019. Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes. Scientific Reports. 9:3127. https://doi.org/10.1038/s41598-019-39753-7.
Huynh, M.P., Bernklau, E.J., Coudron, T.A., Shelby, K., Bjostad, L.B., Hibbard, B.E. 2019. Characterization of corn root factors to improve artificial diet for western corn rootworm (Coleoptera: Chrysomelidae) larvae. Journal of Insect Science. 19(2):1-8. https://doi.org/10.1093/jisesa/iez030.
Geisert, R.W., Cheruiyot, D.J., Hibbard, B.E., Shapiro Ilan, D.I., Shelby, K., Coudron, T.A. 2018. Comparative assessment of four steinernematidae and three heterorhabditidae species for infectivity of larval diabrotica virgifera virgifera. Journal of Economic Entomology. 111(2):542-548. https://doi.org/10.1093/jee/tox372.
Bernklau, E.J., Hibbard, B.E., Bjostad, L.B. 2018. Repellent effects of methyl anthranilate on western corn rootworm lLarvae (Coleoptera: Chrysomelidae) in soil bioassays. Journal of Economic Entomology. 112(2):683-690. https://doi.org/10.1093/jee/toy346.
Vanous, A., Gardner, C.A., Blanco, M., Martin-Schwarze, A., Wang, J., Li, X., Lipka, A.E., Flint Garcia, S.A., Bohn, M., Edwards, J.W., Lübberstedt, T. 2018. Stability analysis of kernel quality traits in exotic-derived doubled haploid maize lines. The Plant Genome. 12(1). https://doi.org/10.3835/plantgenome2017.12.0114.
Rathnayake, K.N., Nelson, S.K., Seeve, C.M., Oliver, M.J., Koster, K. 2018. Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance. Physiologia Plantarum. https://doi.org/10.1111/ppl.12892.
Yobi, A., Batushansky, A., Oliver, M.J., Angelovici, R. 2019. Adaptive responses of amino acid metabolism to the combination of desiccation and low nitrogen availability in Sporobolus stapfianus. Planta. 249(5):1535-1549. https://doi.org/10.1007/s00425-019-03105-6.
Alkhalifah, N., Campbell, D., Falcon, C., Miller, N., Romay, M., Walls, R., Walton, R., Yeh, C., Bohn, M., Buckler IV, E.S., Ciampitti, I., Flint Garcia, S.A., Gore, M., Graham, C., Hirsch, C., Holland, J.B., Hooker, D., Kaeppler, S., Knoll, J.E., Lauter, N.C., Lee, E., Lorenz, A., Lynch, J., Moose, S., Murray, S., Nelson, R., Rocheford, T., Rodriguez, O., Schnable, J., Scully, B.T., Smith, M., Springer, N., Thomison, P., Tuinstra, M., Wisser, R., Xu, W., Ertl, D., Schnable, P., De Leon, N., Spalding, E., Edwards, J.W., Lawrence-Dill, C. 2018. Maize genomes to fields: 2014 and 2015 field season genotype, phenotype, environment, and inbred ear image datasets. Biomed Central (BMC) Plant Biology. 11:452. https://doi.org/10.1186/s13104-018-3508-1.