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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

2017 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
Objective 1. We have completed all phenotypic and genotypic activities for the Zea Synthetic doubled haploid (DH) population comprised of nearly 2000 DH lines ahead of schedule. 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 have completed multiple rounds of seed increase, but continue to attempt increases of problematic lines for the public release that is scheduled at the end of FY17. We have also completed all phenotypic and genotypic activities for the Teosinte Synthetic population ahead of schedule. The analysis has been completed and a draft manuscript has been prepared for publication. Objective 2. The kernel composition fine mapping project (1.4) has been delayed by two consecutive previous crop failures. Initial crosses have been made for a small number of near isogenic lines (NILs), and the F1s have been self-pollinated to create F2 populations. Objective 3. We completed construction of artificial micro-ribonucleic acids (miRNAs) under the control of a chemically inducible promoter to control timing of expression and once the first set of transgenics (T0) are available we will initiate testing of the efficacy of the constructs and the inducibility of the promoter. We have extended the detailed comparison of nitrogen remobilization during dehydration stress, comparing plants with added nitrogen and low nitrogen inputs to include all tissues within the plant and completed a protein profile of the older mature leaves. The genomes of both grass models for plant dehydration aspects of the plan are now complete and annotated. These genomes serve as a valuable genomic resource and enabled us to ensure that we can target the specific member of the gene family that controls and regulates the biosynthesis and catabolism of our target metabolites, in S. stapfianus and by extension maize. We have mapped the transcriptomes to the genome and we are now targeting the genes for our transgenic strategy of functional analysis related to drought tolerance. We are also exploring a separate strategy using seed biology to establish the functionality of the target genes in dehydration tolerance. Objective 3. We have initiated a sequencing of the genome for the maize line that is currently targeted for the nodal root studies aimed at understanding the genomic and physiological root growth responses to water deficit stress in maize (sub-ordinate project 5070-21000-038-09R). The sequences generated from a high-throughput next generation sequencing strategy will be assembled using the B73 reference genome and 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 experiments to feed into the experimental “omics” pipelines for data and hypothesis generation. Objective 5: We have fully developed the soil-plate based system for quantifying wheat seedling root length in under water stress conditions and have screened some of the available genetic diversity for water deficit responses of roots of wheat. We have identified contrasting genotypes that differ in their root growth responses and we are exploring the extent of this variation using different stress treatments. We are also continuing to screen wheat genotypes and wheat mutant lines, deficient in hormone signaling pathways, that we have obtained from ARS in Pullman Washington. We have 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) and refined the protocols to allow for the quantification of ABA and several GA metabolites in a single run. We have collected tissues for hormone extractions from seminal roots of several wheat varieties under water stress. The first analyses of these extracts indicate some important variation in hormone levels that we are currently assessing and validating. We have also initiated plans to build a root phenotyping “robot” system to allow for the high throughput analysis of water deficit stress responses of wheat roots (and other species), not only to speed up our analyses but also in preparation for GWAS genetic analyses that we are planning for the next project plan cycle. Objectives 6 and 7: For the base project, we have begun research with the northern corn rootworm. At this point, we have evaluated five public diets available for the western corn rootworm larvae to evaluate growth and development of the northern corn rootworm colony from USDA-ARS, Brookings, South Dakota. Survivorship was near 100% for three of the diets for 10 days. Dry weight of northern corn rootworm larvae from the USDA-ARS diet, MO-2015 was more than double the dry weight of larvae from the other two diets supporting survivorship. Molting occurred on two of the diets. Nearly 100% of larvae molted within 10 days on the MO-2015 diet while less than 80% molted on the Pleau et al. (2002) diet. No larvae molted on the other diets evaluated. Additional experiments are underway toward an improved diet specialized for northern corn rootworm larvae. In addition, we have evaluated oviposition of wild northern corn rootworm beetles under differing female densities and oviposition media. Nearly 100 fold more eggs were laid per female in small cages with single females than from larger cages with 25, 100, or 500+ females. Oviposition media mattered far less than female density. The data suggest the possibility of an oviposition deterring pheromone being deposited with the eggs and we intend to investigate this hypothesis further in future work. Finally, we are in the process of evaluating northern corn rootworm for baseline susceptibility to current single Bacillus thuringiensis (Bt) toxins. Objective 7: With our western corn rootworm colonies, we continue to select for survivorship on Bt corn to expressing Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1 toxins. This year, we were first to report resistance to Cry34/35Ab1 in diet toxicity assays. A 40-fold increase in LC50 values were documented in our Cry34/35Ab1-selected colonies compared to our control colonies. This still was not full resistance. Larvae from the selected colonies were still significantly smaller on Cry34/35Ab1 than non-Bt corn with the same genetic background. In addition, the project continues to develop germplasm/populations and statistical methods related to the analysis of quantitative traits and population genetics. 1) We performed greenhouse crossing to complete the development of our epistasis mapping population (EMP). A preliminary trial of EMP germplasm was conducted summer 2017. We conducted simulations to evaluate the power of EMPs for mapping epistatic interactions. 2) We continued our long-term selection program for plant height in the Shoepeg maize population. Samples were phenotyped and genotyped and a genome-wide association study was performed. Results are in preparation for publication. The second generation of selections was performed summer 2017. 3) We developed a technique to test for associations between entire biochemical pathways and phenotypes. We applied this technique to amino acid traits. 4) We finished developing a method to test whether or not particular quantitative traits have been selected over the course of the past several generations. Inputs for this method are a modern genotyped and phenotyped population, coupled with an estimate of allele frequencies some number of generations in the past. This test can inform breeders about which traits have been historically important and which they should continue to breed for in the future. 5) We continued to develop a NIL population based on the cross of the inbred line B73 and the inbred line B97.

1. High throughput system to visibly monitor root growth under water deficit stress. Drought threatens food security and contributes to the growing problem of malnutrition and hunger. Because of their role in supplying water and minerals to the plant, roots are at the center of the response to water deficit and the signals they generate are critical to the overall plant’s response to a dehydration event. ARS scientists in Columbia, Missouri have developed a system that allows researchers to digitally monitor root growth responses in crops, in particular wheat and corn, to low soil moisture contents. ARS researchers have screened for wheat varieties that have roots that better maintain active growth under water deficit (drought) conditions. The rapid analysis of root growth will enable the development of novel breeding strategies to improve the drought tolerance of economically important crops.

2. Improved artificial diet for western corn rootworm bioassays. Insect resistance to transgenic crops expressing one or more genes from Bacillus thuringiensis (Bt) is a growing concern for farmers and researchers alike. Western corn rootworm is a pest of corn in the United States of America and is targeted by four different Bt proteins. Since 2009, instances of field-evolved Bt resistance have been documented for each Bt protein targeting western corn rootworm. Researchers at Columbia, Missouri recently developed an improved diet for western corn rootworm larvae. In addition to the nutritional improvements, they have tested the compatibility of all four marketed Bt proteins for the control of western corn rootworm with a susceptible colony on our diet and the proprietary diet typically used for each toxin. The diet is compatible with each of the proteins and can differentiate resistant strains from susceptible strains for each protein. As a result, researchers will have the ability directly compare toxins targeting this major pest for the first time.

Review Publications
Beissinger, T.M., Morota, G. 2017. Medical subject heading (MeSH) annotations illuminate maize genetics and evolution. Plant Methods. 13:8. doi: 10.1186/s13007-017-0159-5.
Walsh, J.R., Schaeffer, M.L., Zhang, P., Rhee, S.Y., Dickerson, J.A., Sen, T.Z. 2016. The quality of metabolic pathway resources depends on initial enzymatic function assignments: a case for maize. BMC Systems Biology. 10:129. doi: 10.1186/s12918-016-0369-x.
Gu, X., Lee, T., Geng, T., Liu, K., Thoma, R., Crowley, K., Edrington, T., Ward, J., Wang, Y., Flint Garcia, S.A., Bell, E., Glenn, K. 2017. Assessment of natural variability of maize lipid transfer protein using a validated sandwich ELISA. Journal of Agricultural and Food Chemistry. 65:1740-1749. doi: 10.1021/acs.jafc.6b03583.
Yobi, A., Schlauch, K.A., Tillet, R.L., Yim, W.C., Espinoza, C., Wone, B.W., Cushman, J.C., Oliver, M.J. 2017. Sporobolus stapfianus: Insights into desiccation tolerance in the resurrection grasses from linking transcriptomics to metabolomics. Biomed Central (BMC) Plant Biology. 17:67.
Costa, M.C., Farrant, J.M., Oliver, M.J., Ligterink, W., Buitink, J., Hilhorst, H.M. 2016. Key genes involved in desiccation tolerance and dormancy across life forms. Plant Science. 251:162-168. doi: 10.1016/j.plantsci.2016.02.001.
Hiltpold, I., Hibbard, B.E. 2016. Neonate larvae of the specialist herbivore Diabrotica virgifera virgifera do not exploit the defensive volatile (E)-ß-caryophyllene in locating maize roots. Journal of Pesticide Science. 89(4):853-858. doi:10.1007/s10340-015-0714-7.
Lennon, J., Krakowsky, M.D., Goodman, M., Flint Garcia, S.A., Balint Kurti, P.J. 2017. Identification of Teosinte (Zea mays ssp. parviglumis) alleles for resistance to southern leaf blight in near isogenic maize lines. Crop Science. doi:10.2135/cropsci2016.12.0979.
Liu, Z., Garcia, A., McMullen, M., Flint Garcia, S.A. 2016. Genetic analysis of kernel traits in maize-teosinte introgression populations. G3, Genes/Genomes/Genetics. 6(8):2523-2530. doi: 10.1534/g3.116.030155/-/DC1.
Bernklau, E.J., Hibbard, B.E., Norton, A., Bjostad, L.J. 2016. Methyl anthranilate as a repellent for western corn rootworm larvae (Coleoptera: Chrysomelidae). Journal of Economic Entomology. 109(4):1683-1690. doi: 10.1093/jee/tow090.
Geisert, R.W., Hibbard, B.E. 2016. Evaluation of potential fitness costs associated with eCry3.1Ab resistance in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Economic Entomology. 109(4):1853-1858.
Ludwick, D.C., Meihls, L.N., Ostlie, K.R., Potter, B.D., French, L., Hibbard, B.E. 2017. Minnesota field population of western corn rootworm (Coleoptera: Chrysomelidae) shows incomplete resistance to Cry34Ab1/Cry35Ab1 and Cry3Bb1. Journal of Applied Entomology. 141:28-40. doi: 10.1111/jen.12377.
Karn, A., Heim, C., Flint Garcia, S.A., Bilyeu, K.D., Gillman, J.D. 2017. Development of rigorous fatty acid near-infrared spectroscopy quantitation methods in support of soybean oil improvement. Journal of the American Oil Chemists' Society. 94:69-76. doi:10.1007/s11746-016-2916-4.
Costa, M.D., Artur, M.S., Maia, J., Jonkheer, E., Derks, M.F., Nijveen, H., Williams, B., Mundree, S.G., Jimenez-Gomez, J.M., Hesselink, T., Schijlen, E.G., Ligterink, W., Oliver, M.J., Farrant, J.M., Hilhorst, H.W. 2017. A footprint of desiccation tolerance in the genome of Xerophyta viscosa. Nature Plants. 3:17038. Available:
Karn, A., Gillman, J.D., Flint Garcia, S.A. 2017. Genetic analysis of teosinte alleles for kernel composition traits in maize. G3, Genes/Genomes/Genetics. 7(4):1157-1164. doi:org/10.1534/g3.117.039529.
Seeve, C.M., Cho, I., Hearne, L.B., Srivastava, G.P., Joshi, T., Smith, D., Sharp, R.E., Oliver, M.J. 2017. Water deficit-induced changes in transcription factor expression in maize seedlings. Plant Cell and Environment. 40:686-701. doi: 10.1111/pce.12891.
Bernklau, E.J., Hibbard, B.E., Bjostad, L.J. 2016. Toxic and behavioral effects of free fatty acids on western corn rootworm (Coleoptera: Chrysomelidae) larvae. Journal of Applied Entomology. 140(10):725-735. doi:10.1111/jen.12312.
Qu, W., Robert, C., Erb, M., Hibbard, B.E., Paven, M., Gleede, T., Riehl, B., Kersting, L., Cankaya, A.S., Kunert, A.T., Xu, Y., Schueller, M.J., Shea, C., Alexoff, D., Lee, S.J., Fowler, J.S., Ferrieri, R.A. 2016. Dynamic precision phenotyping reveals mechanism of crop tolerance to root herbivory. Plant Physiology. 172:776-788.
Zukoff, S.N., Zukoff, A.L., Geisert, R.W., Hibbard, B.E. 2016. Western corn rootworm (Coleoptera: Chrysomelidae) larval movement in eCry3.1Ab+mCry3A seed blend scenarios. Journal of Economic Entomology. 109(4):1834–1845.