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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Research Project #424655

Research Project: Genetics and Genomics of Complex Traits in Grain Crops

Location: Plant Genetics Research

2018 Annual Report

Objective 1: Create novel genetic resources for complex trait dissection in diverse maize germplasm. • Sub-objective 1.1: Create, genotype, and phenotype doubled haploid (DH) lines from a synthetic population containing diverse germplasm, including teosinte alleles. • Sub-objective 1.2: Create, genotype and phenotype novel quantitative trait loci (QTL) populations derived from a (teosinte x B73) x B73 population. Objective 2: Characterize the genetic basis of important agronomic traits (heterosis, drought tolerance, yield components, DIMBOA synthesis, and kernel composition) in maize. • Sub-objective 2.1: Determine the genetic basis of heterosis and its relationship to recombination and the Hill-Robertson effect. • Sub-objective 2.2: Fine-map the regulatory site for the major QTL of DIMBOA synthesis for chromosome 4 from CI31A. • Sub-objective 2.3: Fine map the genes responsible for a KRN QTL on chromosome 2 in a teosinte x maize population. • Sub-objective 2.4: Determine the genetic basis of kernel composition in maize x teosinte introgression libraries, and compare the QTL and effects to those observed in maize. Objective 3: Determine molecular and biochemical mechanisms of drought tolerance in maize and model species. • Sub-objective 3.1: Determine the expression patterns of transcription factor (TF) genes in the drought response of maize. • Sub-objective 3.2: To fully characterize the molecular genetic basis of the conserved interplay between reactive oxygen species (ROS) and amino acid metabolism, linked through gamma-glutamyl amino acids (GGAA) metabolism and transport, and the role of GGAA metabolism in dehydration tolerance. Objective 4: Identify and curate key datasets that will serve to benchmark genomic discovery tools for key agronomic traits, especially response to biotic and abiotic environmental stressors. • Sub-objective 4.1: Bring into The Maize Genome Database (MaizeGDB) the phenotypic data generated by critically important research endeavors including the Maize Diversity Project. • Sub-objective 4.2: Curate maize metabolism and pathways data for release as a BioCyc database and as GO annotation files. Objective 5: Characterize the relationship between root biology and drought tolerance in wheat and related species. • Sub-objective 5.1: Elucidate the physiological basis of root growth responses in wheat (hard and soft red winter) and the “wheat model” Brachypodium distachyon, to imposed water deficits. Objective 6: Develop and improve sources of resistance in maize to corn rootworm larval feeding. Objective 7: Characterize Western corn rootworm colonies with resistance to Bacillus thuringiensis (Bt) toxins to facilitate better resistance management decisions.

Create and fully describe double haploid lines and QTL populations for complex trait dissection. Map and characterize yield QTLs to interrogate the genetic basis of heterosis in maize. Use QTL fine mapping protocols to define the genetic regulation of DIMBOA synthesis in maize. Develop targeted metabolomic profiles to define the role of nitrogen metabolism in establishing dehydration tolerance in the C4 grasses, including maize. Combine field experiments and transgenic maize lines to determine the role of selected transcription factors in the response of roots to water deficits and their possible role in drought tolerance. Use modern curation tools to improve the phenotype to gene utility of the MaizeGDB and improve linkages to other community database efforts.

Progress Report
Over the lifetime of the project, this research addressed the National Program 301 Action Plan Component 1 – Crop Genetic Improvement, Problem Statement 1B: Innovative approaches to crop genetic improvement and trait analysis, as well as National Program 304 Action Plan Component 3A – Insects and Mites, Problem Statement 3A2: Systems approach to environmentally sound pest management. In Objective 1, two novel resources were created for dissecting complex traits in diverse maize; the Zea Synthetic doubled haploids (DH) and the Teosinte Synthetic, both of which contain levels of teosinte, the wild ancestor of maize. We have completed nearly all aspects of the Zea Synthetic DH project, including development of the DH resource comprised of nearly 2000 DH lines, seed increase, and both phenotypic and genotypic analyses. The DH were evaluated at 6 locations (Missouri, New York, North Carolina, and Iowa) in 2014 and/or 2015 for maturity, plant height, ear traits, and kernel traits. We are currently conducting the final round of seed increase of problematic lines for the public release via the Maize Genetics Stock Center at the end of FY18. Genetic analysis is ongoing including genome wide association mapping (GWAS) of all traits and the analysis of haplotypes that have been selected against during DH development. We are in the process of reanalyzing the genotypic data based on allele frequencies rather than genotypic calls, but a draft manuscript has been written as part of a student dissertation. We have also made hybrid seed of approximately 500 of the DH, and a yield trial is underway in Missouri in summer 2018. We have also completed all phenotypic and genotypic activities for the Teosinte Synthetic project. Preliminary analyses have been completed and a draft manuscript has been prepared for publication, but a final analysis is underway prior to submission. Objective 3 of this project addressed the complex trait of drought tolerance in maize and we completed a full analysis of the drought response genetic controlling elements, transcription factors (TFs) and miRNAs (small molecules that control gene expression). We identified over 400 TFs that respond to drought in various tissues of the maize plant. We have focused on over 100 transcription factors that respond in the primary root tip and that potentially control root growth under drought conditions. We completed construction of artificial micro-ribonucleic acids (miRNAs) under the control of a chemically inducible promoter to control timing of expression of a number of these transcription factors and we are awaiting the modified seed from the transformation facility. We also generated a network analysis of co-expressed genes controlled by the TFs and key processes that relate to root growth under soil water deficits have been revealed for further research efforts. The root tip responsive TFs form the basis of one of the objectives in our new OSQR approved project plan. We also completed a detailed analysis of the role of miRNAs in the maize root response to soil water deficits and have demonstrated a link between drought tolerance and phosphate metabolism through the expression of a “target mimic” small RNA that acts to suppress a specific miRNA that controls phosphate uptake. We fully characterized the response of two sister species of grass (a model system), one desiccation tolerant the other desiccation sensitive, to reveal genes and gene networks associated with dehydration tolerance. We have completed the detailed comparison of nitrogen remobilization throughout the whole plant and its link to oxidative metabolism during dehydration stress, comparing grasses grown with added nitrogen and low nitrogen. We have identified genes involved in the biosynthesis of gamma-glutamyl dipeptides, a targeted metabolite from our earlier studies to manipulate desiccation tolerance in a model species to assess efficacy for use in maize. We have completed a proteomics study of leaves during dehydration to further evaluate potential targets for drought tolerance improvement efforts. The two genomes for the grasses have been sequenced and all transcriptomic bioinformatic analyses have been completed. We are identifying key control genes in the dehydration tolerance gene networks that will be targeted for analysis in maize to improve drought tolerance in our new project plan. In addition to our Project Plan, we have initiated a sequencing of the full-length transcriptome (exome) of the maize line that is currently targeted for the nodal root studies to add to our assembled genome and to generate a read mapping resource for expression transcriptome studies (sub-ordinate project 5070-21000-038-09R). This will enable the group to fully take advantage of the planned transcriptomic and metabolomics data we will generate. We are currently collecting the necessary root samples from both lab and field based metabolomic and proteomic pipelines for data and hypothesis generation. Objective 5 related to drought tolerance in wheat and over the life time of the project we developed a soil-plate based system for quantifying wheat seedling root length in under water stress conditions and identified contrasting genotypes that differ in their root growth responses and we have measured the extent of this variation using different stress treatments and a range of wheat varieties. We are currently screening wheat mutant lines, deficient in hormone signaling pathways, obtained from ARS in Pullman Washington. We developed a method for extraction and quantification of Abscisic Acid (ABA) and Giberellic Acid (GA) hormones from wheat roots by liquid chromatography-mass spectroscopy (LC-MS) in a protocol that quantifies the target hormones in a single run. We have observed important variation in hormone levels that we are continuing to assess and validate. We have also identified a unique temperature related phenotype that is revealed when used in combination with a water deficit. We have also completed the construction of a root phenotyping “robot” system to allow for the high throughput analysis of water deficit stress responses of wheat roots (and other species). This system is currently being tested and will be central piece of apparatus for achieving our new objectives. In Objectives 6 and 7, we developed western corn rootworm colonies with resistance to all current Bt toxins targeting this major pest and documented specific levels of resistance and cross resistance associated with these colonies. We have developed a new artificial diet for western corn rootworm larvae and documented that it is compatible with all current Bt toxins. We have evaluated hundreds of maize lines for natural resistance to feeding damage from western corn rootworm larvae and recently reported genetic markers associated with natural resistance to larval feeding.

1. Genome and genomic resources for dehydration tolerance. Drought threatens food security and contributes to the growing problem of malnutrition and hunger. To date researchers have yet to uncover the fundamental and adaptive aspects of dehydration tolerance, an important component for maintaining physiological activity under severe drought stress, and that ultimately underpins the ability to survive in drying soils. This is because crops, and their ancestors, have little tolerance to cellular dehydration. ARS researchers in Columbia, Missouri, have sequenced the complete genomes of two grass species, often used as forage, that differ considerably in their ability to tolerate dehydration. Sporobolus stapfianus can survive the complete desiccation of its vegetative tissue whilst, Sporobolus pyramidalis like most grasses, including those that constitute major crops in the U.S., can only survive a mild dehydration (drought). The two genomes have been fully annotated to mark the position and identity of each gene. The expression characteristics of each gene during dehydration, mild water loss in the case of S. pyramidalis and full dehydration and rehydration for S. stapfianus, have been mapped and measured for both species and in both shoots and roots. The establishment of these genome and genomic resources in two closely related species has identified both gene and gene networks that are adaptive in the dehydration (drought) tolerance mechanisms in the grasses. These gene and gene networks provide genetic targets for manipulation or breeding for improved drought tolerance in grass crops, in particular maize, sorghum, and forage species.

2. Zea Synthetic doubled haploids – A new resource for maize. There is nearly an infinite number of rare gene variants that may play a significant role in complex traits in crops such as maize (corn). Some of these rare variants may be beneficial to agriculture but may not be present in current maize breeding populations. Researchers in Columbia, Missouri created a population of nearly 2000 double haploid (DH) lines from a unique intermated population called the “Zea Synthetic,” comprised of 88% inbred lines from around the world and 12% teosinte, the wild ancestor of corn. The goal of the study was to understand the frequency and strength of negative and beneficial rare variants, and what happens to them in a corn breeding program. The DH lines were evaluated in field trials for agronomic (maturity, plant and ear height, number of ears) and fitness (total kernel number and kernel weight) traits.

Review Publications
Mahmoud, M.A., Sharp, R.E., Oliver, M.J., Finke, D.L., Bohn, M., Ellersieck, M.R., Hibbard, B.E. 2018. Response of maize hybrids with and without rootworm-and drought-tolerance to rootworm infestation under well-watered and drought conditions. Journal of Economic Entomology. 111(1): 193-208.
Flint Garcia, S.A. 2017. Maize kernel evolution: From teosinte to maize. CAB International United Kingdom. In: Larkins, B., editor. Maize Kernel Development. Oxfordshire, UK. CAB International United Kingdom. p. 1-15.
Wang, L., Beissinger, T.M., Lorant, A., Ross-Ibarra, C., Ross-Ibarra, J., Hufford, M.B. 2017. The interplay of demography and selection during maize domestication and expansion. Genome Biology. 18:215.
Ludwick, D.C., Zukoff, A.L., Higdon, M.L., Hibbard, B.E. 2018. Protandry of western corn rootworm (Coleoptera: Chrysomelidae) beetle emergence partially due to earlier egg hatch of males. Journal of Kansas Entomological Society. 90(2):94-99.
Bohn, M.O., Marroquin, J.J., Flint Garcia, S.A., Dashiell, K.E., Wilmot, D.B., Hibbard, B.E. 2018. Quantitative trait loci mapping of western corn rootworm (Coleoptera: Chrysomelidae) host plant resistance in two populations of doubled haploid lines in maize (Zea mays L.). Journal of Economic Entomology. 111(1):435-444.
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.
Gage, J., Jarquin, D., Romay, M., Lorenz, A., Buckler IV, E.S., Kaeppler, S., Alkhalifah, N., Bohn, M., Campbell, D., Edwards, J.W., Ertl, D., Flint Garcia, S.A., Gardiner, J., Good, B., Hirsch, C., Holland, J.B., Hooker, D., Knoll, J.E., Kolkman, J., Kruger, G., Lauter, N.C., Lawrence-Dill, C., Lee, E., Lynch, J., Murray, S., Nelson, R., Petzoldt, J., Rocheford, T., Schnable, J., Schnable, P., Scully, B.T., Smith, M., Springer, N., Srinivasan, S., Walton, R., Weldekidan, T., Wisser, R., Xu, W., Yu, J., De Leon, N. 2017. The effect of artificial selection on phenotypic plasticity in maize. Nature Communications. 8:1348.
Perroud, P., Hass, F., Hiss, M., Ullrich, K., Alboresi, A., Amirebrahimi, M., Barry, K., Bassi, R., Bonhomme, S., Chen, H., Coates, J., Fujita, T., Guyon-Debast, A., Lang, D., Lin, J., Lipzen, A., Nogue, F., Oliver, M.J., Ponce De Leon, I., Quatrano, R.S., Rameau, C., Reiss, B., Reski, R., Ricca, M., Saidi, Y., Sun, N., Szovenyi, P., Sreedasyam, A., Grimwood, J., Stacey, G., Schmutz, J., Rensing, S.A. 2018. The Physcomitrella patens gene atlas project: large scale RNA-seq based expression data. Plant Journal. 95:168-182.
Nelson, S.K., Oliver, M.J. 2017. A soil-plate based pipeline for assessing cereal root growth in response to polyethylene glycol (PEG)-induced water deficit stress. Frontiers in Plant Science. 8:1272.
Xiao, L., Yobi, A., Koster, K., He, Y., Oliver, M.J. 2017. Desiccation tolerance in Physcomitrella patens: Rate of dehydration and the involvement of endogenous abscisic acid (ABA). Plant Cell and Environment. 41:275-284.