Research Project: Characterization of Genetic Diversity in Soybean and Common Bean, and Its Application toward Improving Crop Traits and Sustainable Production

Location: Soybean Genomics & Improvement Laboratory

2020 Annual Report

Objectives
Objective 1: Discover QTL and genes controlling biotic and abiotic stress tolerance, and agronomic and quality traits in soybean and common bean and develop new DNA markers that define haplotype variation across new and previously identified genomic regions. [NP301, C1, PS1A; C3, PS3B] The aim of objective 1 is to develop community resources for efficient identification of genes/QTL impacting a range of traits and to facilitate marker assisted selection of alleles in soybean and common bean in collaboration with breeders. These include highly polymorphic markers, core germplasm collection and genotypic datasets of new exotic elite germplasm introduced to USDA Soybean Germplasm Collection. Objective 2: Evaluate diverse soybean populations developed from hybridization with wild soybean to discover unique QTL controlling seed protein and oil content, develop molecular markers, and make these available to breeders for improving soybean quality. [NP301, C1, PS1A; C3, PS3B] As many wild soybean germplasm may has different alleles controlling high protein and oil content than cultivated soybean, here we will explore wild soybean for the improvement of U.S. soybean seed protein and oil content with the markers developed from Objective 1 and genomic tools previously developed in our laboratory. Objective 3: Characterize genetic diversity of the Soybean Rhizobium Germplasm Collection using whole genome sequencing, evaluate nitrogen fixation efficiency of the core strains, and use the information to identify rhizobium genes associated with host-specific nodulation and nitrogen fixation in specific soybean genotype/rhizobium symbioses. [NP301, C1, PS1A; C3, PS3B] Genetic diversity of the rhizobia will be evaluated using genomic information and their influence on the nitrogen fixation efficiency in soybean will be analyzed. The research will result in the identification of efficient strains and genes for enhanced nitrogen fixation in soybean, resulting in better utilization of the diversity of rhizobium strains and soybean ancestors to improve biological nitrogen fixation in commercial soybean cultivars.

Approach
Objective 1: Solexa short genomic DNA sequences from 16 diverse genotypes of different common bean market classes will be aligned to the common bean whole genome sequence (WGS) for SSR marker discovery. After filtering, primer sets will be designed to amplify the SSRs. A subset of 100 primer pairs will be randomly selected for testing polymorphism using genomic DNA from the 16 diverse common bean genotypes. A total of 12 pairs of diverse genotypes from different market classes of the Andean Diverse Panel of common bean will be sequenced. Called SNPs will be filtered based on a number of factors for beadchip assay. SNPs that are polymorphic within multi- market classes will be added to the Illumina Infinium BARCBean6K_3 BeadChip pool or used for KASP markers to fine map gene/QTL in targeted genomic regions. Based on the SNP data of the >18,000 cultivated soybean accessions assayed with SoySNP50K BeadChip, core sets of soybean accessions for each soybean maturity group will be created. The software Core Hunter 3 will be used to select the core collection with high allelic richness. Objective 2: a nested association mapping panel consisting of 150-300 F6 lines from each of 10 crosses of NC-Raleigh x wild soybean from the wild soybean core collection will be developed. The parents and the RILs will be grown in the field at two locations in two years. DNA isolated from the RILs and parents will be genotyped with Illumina BARCSoySNP6K BeadChips. Protein content and oil content of the parents and lines will be measured using a DA 7250 NIR Analyzer. The dataset will be used to identify QTL, genes and haplotypes controlling high seed protein and oil content in wild soybean that will be used for improving cultivated soybean and to predict accuracy of genomic selection. Objective 3: Genomic DNA of 760 soybean Bradyrhizobium strains will be isolated and sequenced at using NextSeq500 Sequencer. The resulting sequence will be aligned to the WGS of the B. japonicum strain USDA110 for variant discovery. Redundant or highly similar strains with 99.9% similarity among the soybean rhizobia will be identified. Within each cluster with 99.9% similarity, an accession from each cluster will be evaluated for nitrogen fixation efficiency using 8 ancestral cultivars which contribute more than 70% of the genetic diversity to the Southern and Northern American elite cultivars. Plant will be measured for chlorophyll content and biomass with or without inoculation of the stains, and scored for plant vegetative growth based on the growth of the plant inoculated with USDA110, a recommended soybean strain. The test in eight ancestors will be carried out in a greenhouse with replications.

Progress Report
Progress was made to develop core sets of soybean germplasm for different soybean growing regions. Based on the dataset of 18,489 cultivated soybean accessions genotyped with 42,509 SNPs from the BARCSoySNP50K assay, we selected ten sets of soybean accessions that can adapt to each soybean region and that capture at least 95% of the diversity of the SNPs among the accessions from the region. Each core set contained 100-500 accessions depending on the number of available soybean accessions adapted to the regions, the genetic diversity among the accessions from the region, and breeders’ capacity to evaluate the phenotypes. Some of the core sets have been provided to soybean breeders and geneticists working at different regions, e.g. the core set of 500 accessions for maturity group (MG) II, III and IV was provided to Purdue University, the core set of 500 from MG IV and V was provided to Virginia Tech, the core set of 300 from MG IV,V,VI, VII was provided to USDA ARS, Raleigh, North Carolina, the core set of 500 from MG V, VI, VII, VIII and IX was provided to the University of Georgia. We also provided 200 most diverse soybeans from MG000-X to USDA-ARS, Jackson, Tennessee, and 400 accessions from MG000-X to the University of Missouri. In addition, based on the dataset of 1,168 G. soja accessions genotyped with the 42,509 SNPs, we selected a set of 400 wild soybeans from MG III and IV for USDA ARS, Stoneville, Mississippi, and a core set of wild soybean accessions from MG000-X for the University of Missouri and USDA ARS, Raleigh, North Carolina. These core collections of the soybean are critical for the breeders and geneticists to efficiently evaluate and utilize the large number of accessions in the collection for the discovery of novel genes/quantitative trait loci (QTL) controlling important traits. Progress was made to advance populations from the crosses between cultivated soybean and wild soybean to discover unique quantitative trait loci (QTL) controlling seed protein and oil content in wild soybean, to develop molecular markers, and to make these available to breeders for improving soybean quality. Using the single seed descent method, a total of 10 G. max x G. soja families (NC-Raleigh × PI 549032, NC-Raleigh × PI378684B, NC-Raleigh × PI378690, NC-Raleigh × PI378696B, NC-Raleigh × PI407020, NC-Raleigh × PI407228, NC-Raleigh × PI424007, NC-Raleigh × PI424045, NC-Raleigh × PI424083A, and NC-Raleigh × PI562551) with a common G. max NC-Raleigh parent were grown at Beltsville, Maryland, and Raleigh, North Carolina, in 2019. Field experiments of 1,005 lines were conducted with a randomized block design of two replications at Beltsville and a complete randomized design of one replication at Raleigh. Genotyping of 1,005 RILs, their parents and controls with the BARCSoySNP6K assay containing 6,000 SNPs was completed. The seeds from >3,000 plots harvested at two locations were measured for quality traits including protein, oil content, amino acids and fatty acids using a DA 7250 near-infrared analyzer. Progress was made in the characterization of genetic diversity of the USDA Rhizobium Germplasm Collection using whole genome sequencing. About 550 soybean Bradyrhizobium strains have been grown and isolated from cultured cells, and genomic DNA for approximately 240 strains has been extracted. The DNA of these isolates will be sequenced once the DNA from other isolates are completed. Progress was made to characterize new elite soybean germplasm introduced to the USDA ARS Soybean Germplasm Collection. The Soybean Genomics and Improvement Laboratory at Beltsville, Maryland, previously genotyped over 20,000 soybean accessions in the Collection with >50,000 single nucleotide polymorphisms (SNPs) markers. This information is in Soybase (https://soybase.org/snps/download.php) and is being used to mine genes/alleles controlling seed quality, resistance to abiotic- and biotic-stresses, etc., and has assisted more than 400 publications. Since the genotyping project was completed, new elite accessions were introduced from other countries into the USDA Soybean Germplasm Collection. In collaboration with the scientists at the USDA Soybean Germplasm Collection, we genotyped 576 soybean germplasm accessions from Korea, Vietnam and other countries with the SoySNP50K BeadChips. Thus, the total number of new accessions we have genotyped in FY2019 and FY2020 reached 1,056. The resulting genotypic dataset will be merged with our previous SoySNP50K genotypic dataset and will be deposited at Soybase for public access. We also will compare the genetic relationship of these accessions with the 562 elite cultivars in the USDA-ARS Soybean Germplasm Collection. Those subpopulations that are not represented in current elite cultivars will be provided as a pool of untapped genetic variability that can be exploited for genetic advance for abiotic and biotic stress resistance, seed quality traits, and productivity. Progress was made in the discovery of genes or QTL controlling disease resistance, agronomic and seed composition traits in soybean and common bean. The molecular markers and assay developed by USDA ARS scientists at Beltsville, Maryland, were used to analyze soybean and common bean genetic populations created by collaborators across the U.S. and other countries. The analyses resulted in mapping genomic regions or genes controlling numerous soybean traits including resistance to sudden death syndrome and cyst nematode, seed compositions and agronomic traits in collaboration with researchers in Michigan State University, University of Tennessee, University of Missouri, and Hebei Academy of Agricultural Science in China. For common bean, new germplasm or candidate genes controlling Fusarium oxysporum resistance, and pod and seed size also were identified in collaboration with researchers at Regional Agrifood Research and Development Service (SERIDA) in Spain, and the University of Nova de Lisboa in Portugal.

Accomplishments
1. Three new reference-quality genome assemblies for soybean. Plant breeders and scientists use plant DNA genome sequences to identify genes that are important for yield, disease resistance, and plant growth characteristics. For soybean, a genome sequence assembly for the widely-used Williams 82 variety has been available for about ten years, but there were numerous known gaps and errors in the sequence assembly. USDA-ARS scientists at Beltsville, Maryland, and researchers at the Department of Energy Joint Genome Institute, Hudson-Alpha Institute for Biotechnology, University of Missouri, Iowa State University, University of Minnesota, University of Georgia, University of Australia, Chinese University of Hong Kong, and The International Crops Research Institute for the Semi-Arid Tropics (CRISAT), India, made major improvements to the Williams 82 reference genome sequence, as well as built new genome assemblies for two other soybean varieties: one for the most important U.S. southern cultivar 'Lee', and another for the closest wild relative of soybean, Glycine soja. These three genome sequences are available to the public and can be used to identify similarities and differences that underpin important agricultural traits.

2. Soybean cyst nematode resistance gene function. Soybean cyst nematode (SCN) is the most damaging soybean pathogen, causing significant yield and quality losses. Resistance to SCN is conferred by genes at two main soybean DNA regions, Rhg1 and Rhg4, at chromosome 18 and 8, respectively. The molecular mechanisms through which Rhg1 mediates SCN resistance is known, but the function of Rhg4 remains to be elucidated. The Rhg4 locus contains only one gene encoding serine hydroxymethyltransferase (GmSHMT08). USDA-ARS scientists at Beltsville, Maryland, and researchers at University of Tennessee and Southern Illinois University studied the function of GmSHMT08 in establishing DNA methylation landscapes of soybean roots during SCN infection. They observed that the susceptible soybean exhibited reduced DNA methylation levels in both protein-coding genes and transposable elements, whereas the resistant line showed the opposite response in response to SCN infection. This trend was observed in all DNA methylation contexts, suggesting that GmSHMT08's function is vital for cellular methyltransferase activity, possibly regulating soybean DNA through methylation. Scientists at universities, government agencies, and private institutes will be able to use this knowledge to help shape soybean defense responses upon SCN infection.

Review Publications
Qin, J., Shi, A., Song, Q., Li, S., Wang, F., Cao, Y., Ravelombola, W., Yang, C., Zhang, M. 2019. Genome wide association study and genomic selection of amino acid concentrations in soybean seeds. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.01445.
Murube, E., Campa, A., Song, Q., Mcclean, P., Ferreira, J.J. 2019. Toward validation of QTLs associated with pod and seed size in common bean using two nested recombinant inbred line populations. Molecular Breeding. 40:7. https://doi.org/10.1007/s11032-019-1085-1.
Leitao, S., Malosetti, M., Song, Q., Eeuwijk, F., Rubiales, D., Patto, M. 2020. Natural variation in Portuguese common bean germplasm reveals new sources of resistance against Fusarium oxysporum f. sp. phaseoli and resistance-associated candidate genes. Phytopathology. 110(3):663-647.
Rambani, A., Pantalone, V., Yang, S., Rice, H., Song, Q., Mazarei, M., Arelli, P.R., Meksem, K., Stewart, N., Hewezi, T. 2020. Identification of introduced and stably inherited DNA methylation variants in soybean associated with soybean cyst nematode parasitism. New Phytologist. 227(1):168-184. https://doi.org/10.1111/nph.16511.
Beche, E., Gillman, J.D., Song, Q., Nelson, R.L., Beissinger, T., Decker, J., Shannon, G., Scaboo, A.M. 2020. Nested association mapping of important agronomic traits in three interspecific soybean populations. Theoretical and Applied Genetics. 133:1039-1054. https://doi.org/10.1007/s00122-019-03529-4.
Ma, G.J., Song, Q., Underwood, W., Zhang, Z.W., Fiedler, J.D., Xuehui, L., Qi, L. 2019. Molecular dissection of resistance gene cluster and candidate gene identification of Pl17 and Pl19 in sunflower by whole-genome resequencing. Scientific Reports. 9:14974. https://doi.org/10.1038/s41598-019-50394-8.
Valliyodan, B., Cannon, S.B., Bayer, P.E., Shu, S., Brown, A.V., Ren, L., Jenkins, J., Chung, C.Y.L., Chan, T.F., Daum, C.G., Plott, C., Hastie, A., Baruch, K., Barry, K.W., Huang, W., Gunvant, P., Varshney, R.K., Hu, H., Batley, J., Yuan, Y., Song, Q., Stupar, R.M., Goodstein, D.M., Stacey, G., Lam, H.M., Jackson, S.A., Schmutz, J., Grimwood, J., Edwards, D., Nguyen, H.T. 2019. Construction and comparison of three new reference-quality genome assemblies for soybean. Plant Journal. 100(5):1066-1082. https://doi.org/10.1111/tpj.14500.
Islam, N., Stupar, R., Luthria, D.L., Garrett, W.M., Stec, A.O., Roessler, J., Natarajan, S.S. 2019. Genomic changes and biochemical alterations of seed protein and oil content in a subset of fast neutron induced soybean mutants. Biomed Central (BMC) Plant Biology. 19(1):420. https://doi.org/10.1186/s12870-019-1981-x.
Tan, R., Collins, P.J., Wang, J., Wen, Z., Boyse, J.F., Laurenz, R., Gu, C., Jacobs, J.L., Song, Q., Chilvers, M.I., Wang, D. 2018. Different loci associated with root and foliar resistance to sudden death syndrome (Fusarium virguliforme) in soybean. Theoretical and Applied Genetics. 132:501-513. https://doi.org/10.1007/s00122-018-3237-9.
La, T., Large, E., Taliercio, E.W., Song, Q., Gillman, J.D., Xu, D., Nguyen, H., Shannon, G., Scaboo, A. 2018. Characterization of a USDA core collection of wild soybean (Glycine soja Siebold & Zucc.) accessions for seed composition and agronomic traits. Frontiers in Plant Science. https://doi.org/10.2135/cropsci2017.08.0514.