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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Soybean Genomics & Improvement Laboratory » Research » Research Project #444466

Research Project: Characterization and Utilization of Genetic Diversity in Soybean and Common Bean and Management and Utilization of the National Rhizobium Genetic Resource Collection

Location: Soybean Genomics & Improvement Laboratory

2024 Annual Report


Objectives
Objective 1: Discover genomic loci controlling nutritional quality, stress tolerance, seed yield and other economically important agronomic traits in soybean and common bean and develop genomic tools to enable rapid characterization of populations and selection in breeding programs. Sub-objective 1.A.: Develop efficient genome-wide KASP assays for soybean and common bean genetic and genomic research. Sub-objective 1.B: Discover unique QTL and haplotypes associated with seed methionine content and cysteine content in populations derived from wild and cultivated soybean crosses and evaluate the efficiency of genomic selection for the traits. Discover genomic loci controlling other nutritional quality, stress tolerance, seed yield in soybean and common bean by collaborative research. Objective 2: Identify novel genes controlling rhizobium nodulation in soybean accessions from the National Soybean Germplasm Collection and/or previously reported accessions and determine their underlying mechanism through mapping and gene-structure comparisons. Objective 3: Distribute, acquire and maintain the safety, genetic integrity, and viability of rhizobium genetic resources and associated descriptive information. Objective 4: Conduct research to develop genetic resource maintenance, evaluation, or characterization methods and then apply them to priority rhizobium genetic resources to avoid backlogs in microbial genetic resource and information management. Sub-objective 4.A: Sequencing the rhizobium accessions isolated from the soybean, common bean, and other major legume crops. Sub-objective 4.B: Application of sequencing information to develop core strain collections and high throughput genotyping assays for rhizobium strain identification.


Approach
Objective 1: Sequence reads of common bean accessions will be aligned to the common bean whole genome sequence assembly. Called SNPs will be filtered based on SNP quality and polymorphism. Sequence flanking each of the remaining SNPs will be retrieved for further screening of their sequence specificity in the genome and will be used to design KASP markers. The dataset containing 32 million SNPs from >1500 soybean accessions will be used for soybean KASP assay design per the protocol described above. A total of 10 RIL families from cultivated x wild soybean cross will be used for the discovery of QTL controlling methionine and cysteine content. The parents and RILs were grown in the field at two locations. DNA of the RILs and parents were genotyped with BARCSoySNP6K Chips and the RILs will be imputed with SoySNP50K markers segregration between parents. Seeds will be ground for amino acid measurement. A genome-wide association analysis will be performed. To further fine-map the major QTL regions associated with the amino acid content, residual heterozygous line populations will be developed from the lines that are heterozygous in the identified major QTL regions. Objective 2: A cross between Williams 82 x VS12-0205 (non-nodulation) will be made to create a large F2 population. DNA from leaf tissues of the parent and progenies will be extracted and genotyped with SoySNP50K assay. When matured, each plant will be dug for nodulation observation. JoinMap 4.0 software will be used to map the locus. To further verify the gene that showed expression level difference, RNA from taproot and root hairs of the plants of each parent will be extracted separately at different days after inoculation. The relative expression levels of the candidate gene will be evaluated by the comparative threshold method. To validate the non-nodulation gene function, we will overexpress the candidate gene in VS12-0205 and knock out the candidate gene in the Williams 82 with the RNAi or CRISPR/Cas9-based genome-editing tools. Objective 3: Rhizobial cultures will be managed by their preservation, quality control and disbursement to ARS customers upon request. Technical information about rhizobia, culturing and symbiosis and advice will be given. Emphasis will be placed on preparing and sending cultures for long-term backup at the NCGRP, Fort Collins, CO. The information on old and new strains will be updated and deposited at the National Rhizobium Database for public access. Objective 4: DNA from rhizobium strains will be isolated from major legume crops and sequenced. The resulting sequence will be aligned to the WGS of B. japonicum strain USDA110, B. Elkanii USDA 61 as well as the Sinorhizobium meliloti strain USDA 1002 for SNPs and indels calls. Core sets of rhizobia will be created for common bean and soybean, respectively. A set of SNPs that can distinguish and classify accessions efficiently will be selected to be included in the high-throughput genotyping assay.


Progress Report
Under Objective 1, in collaboration with researchers at ARS-USDA, St Louis, we analyzed sequence reads from 1,512 soybean accessions, including 1308 cultivated soybean and 203 wild soybean accessions, that were generated from our laboratory or collected from the public domains, and identified >32 million single nucleotide polymorphisms (SNPs) and indels after mapping the sequence reads to the Williams82 v2 whole genome sequence assembly. We further eliminated SNPs that are less likely to have base variations among 1308 cultivated soybeans or with a missing and heterozygotic rate greater than 50%, the remaining 5,191,116 SNPs were used for KASP marker design. The 200 bp sequence flanking each SNP was extracted based on Wm82 v2 genome assembly and three primer sets were designed targeting each SNP using Primer 3 program. We used e-PCR software to further eliminate primer sets with non-specific sequences in the genome. For each SNP site, the primer set with the smallest PCR amplicon product was selected and a total of 1,494,086 primer sets were chosen. Our laboratory is using some markers to identify true hybrids in biparental crosses and to fine map of the genes controlling traits, the marker information will be provided to soybean breeders, and deposited at Soybase for public access after the validation is completed. We also selected and provided a set of 4K SNP markers to researchers at the University of Illinois, the International Institute of Tropical Agriculture (IITA), North Dakota, and University of Minnesota and others, which will be used for genomic selection etc. in an African soybean project which is funded by the Gates Foundation. Progress was made to analyze methionine and cysteine content of the nested association mapping (NAM) population consisting of 10 recombinant inbred line (RIL) families derived from 10 diverse wild soybean accessions crossed to a common cultivated soybean cultivar ‘NC-Raleigh’. The parents and 1107 RILs of the 10 crosses were grown in the field at Beltsville, Maryland, and Raleigh, North Carolina, in previous two years with a total of three replicates at locations previously. We analyzed content of sulfur-containing amino acids including methionine, cysteine, lysine, and threonine of 6,642 seed samples on a DA 7250 NIR Analyzer by collaborating with researchers at the University of Georgia and observed the average and range of methionine content (0.55 0.47-0.65), cysteine content (0.59, 0.47-0.72), lysine content (2.77 2.42-3.16) and threonine content (1.67, 1.45-1.90) in the population. The large variation of the amino acids among lines provides a basis for detecting major genomic regions responsible for high sulfur-containing amino acids and the phenotypic dataset will be used to finely map these regions. In addition, we genotyped >4900 soybean and common bean germplasm and breeding lines developed by breeders with assays developed at our laboratory. The analyses of the genotype and phenotypes resulted in mapping of QTL/genes controlling numerous soybean traits including root morphological traits, drought resistance, cyst nematode resistance, seed protein content and oil content, seed sugar composition, etc., genomic selection of seed yield and composition, development of marker associated with seed test weight, and determination of mechanism controlling cyst nematode resistance in collaboration with researchers in the University of Missouri, Virginia Tech, University of Georgia, Virginia State University, University of Arkansas and USDA-ARS, St. Louis, USDA-ARS, Raleigh, North Carolina, etc. In common bean, the analyses led to the development of SNP mark assays for resistance to beet curly top virus and fine-mapping of genes controlling rust resistance in collaboration with researchers at USDA-ARS, Prosser, Washington, and Beltsville, Maryland. These results were all published in pee-reviewed journals. With the funding from the National Science Foundation, we extracted DNA from 430 edamame accessions and genotyped them with SoySNP50K assays, we also extracted DNA from 440 edamame breeding lines for whole genome sequencing. The data will be shared with the PI at Virginia State University and will be used to detect genomic features, and molecular mechanisms regulating seed filling and seed composition. Under Objective 2, through the screening of >1400 soybean lines from the USDA Soybean Germplasm Collection and breeding programs for the nodulation of different soybean rhizobium strains, we discovered the line VS12-0205, developed at Virginia State University, completely restricting nodulations in the greenhouse after inoculating with rhizobium strains and growing in the field. Our preliminary segregation analysis of the nodulation in F1 and F2 progeny from the cross of VS12-0205 x T201(another non-nodulation accession) showed that the non-nodulation trait of VS12-0205 was controlled by a single recessive gene, but the gene was different from the gene controlling non-nodulation in T201. We sequenced VS12-0205 and T201 with sequence coverage >40x and compared to the sequences of known genes controlling non-nodulation in other two accessions: nod 139, and nn5. The VS12-0205 was determined to contain novel genes/alleles that control non-nodulation. In order to map and identify the gene in VS12-0205, a cross between Williams 82 (normal nodulation) x VS12-0205 (non-nodulation) was made and the hybrid seeds were harvested and verified to be true using KASP markers developed in Objective 1. The hybrid seeds were used to develop a F2 mapping population with >1800 plants. Under Objective 3. All stakeholder requests were filled in a timely manner, over 200 strains were provided to 21 research institutions, non-profit organizations, state and private universities. We provided technological support and advice to stakeholders and customers regarding the best strains for each legume, protocols on media preparation and optimal culture growth conditions. We sent 157 accessions of R. leguminosarum viceae to the USDA ARS National Center for Genetic Resources Preservation, in Fort Collins, Colorado. This will ensure that this precious irreplaceable National Resource will always be available for researchers, inoculant producers, and the U.S. growers of legumes. We conducted collection inventory which ensures these agronomically important and irreplaceable resources will always be available for the benefit of U.S. agriculture, and re-stocked over 300 accessions that needed replenishment or were over 30 years old. We demonstrated rhizobium collection work to visitors from commodity group and public event at USDA Headquarters. Updated the electronic searchable database of the entire collection (https://www.ars-grin.gov/~dbmuaz/cgi-bin/rhy/search.pl ). Under Objective 4. A total of 150 rhizobium strains from common bean, soybean and other legumes were activated for DNA isolation. The DNA of these strains, as well as the DNA of strains extracted in subsequent years, will be sequenced, and the sequence reads from these strains and previously sequenced >500 strains will be jointly analyzed to identify variations among the strains. This information will be used to develop core strain collections and high-throughput genotyping assays for rhizobium strain identification. We also provided NA passport information for some accessions to the Joint Genomic Institute, Department of Energy, which is needed to validate strains for sequencing.


Accomplishments
1. Identified germplasm to improve nitrogen fixation efficiency of soybeans in the U.S. Midwest. Soybean is one of the most important economic crops in the world because it is rich in protein and oil. A unique feature of soybeans and other legumes is root nodulation caused by soil rhizobium bacteria. These bacteria are critical for soybeans to assimilate atmospheric nitrogen into organic compounds, reducing the need for nitrogen fertilizers. There are differences in the nitrogen fixation efficiency among the bacteria strains. In the main soybean producing areas of the Midwest U.S., the rhizobium USDA123 strain has a high incidence of root nodules in field soybeans, but it is competitive and inefficient in nitrogen fixation. A potential solution to this problem is the identification of soybean germplasm that restricts nodulation to USDA123 but prefers high nitrogen-fixing efficient strains such as USDA110. USDA-ARS scientists evaluated root nodule numbers in over 1400 cultivated and wild soybean accessions inoculated separately and/or simultaneously with USDA110 and USDA123 and identified soybean accessions that were restricted to USDA123 but preferred USDA110, and further discovered genomic regions controlling the restriction and preference. In addition, a high-throughput system was developed to characterize nodule number and occupancy. Information from this study will aid in the development of USDA123-restricted cultivars that increase nitrogen fixation efficiency and productivity.

2. Discovery of the new gene and mechanism in soybean for conferring resistance to cyst nematode. Soybean cyst nematode (SCN) is a soil-borne pest and the leading biological cause of reduced soybean seed yield. This pathogen costs the United States more than $1.5 billion annually. Identifying and deploying genetic resistance genes in soybeans is the most effective and economical way to control the economic costs of SCN infestation in the field. In this study, scientists at the University of Missouri and USDA-ARS discovered a new resistance gene, GmSNAP02, that confers a unique resistance pattern to SCN through loss-of-function mutations which increased resistance of genome-edited plants. This demonstrates the immediate impact of using GmSNAP02 as a genome editing target to diversify nematode resistance in commercially available varieties, which will accelerate and enhance soybean resistance to SCN.

3. Genetic markers to select and develop drought-tolerant soybean varieties. Drought stress is the leading cause of yield loss in soybean. The obvious solution is irrigation, but only 10% of soybean fields are irrigated. A more sustainable option is to develop drought-tolerant soybean lines. Slow or delayed canopy wilting due to drought stress has been observed in a few exotic soybean accessions and may contribute to yield improvement under drought conditions, however, evaluation of drought stress is time-consuming, expensive and requires a controlled environment. Scientists at University of Georgia and USDA-ARS, Beltsville, Maryland, evaluated different soybean lines derived from drought-resistant exotic soybean accessions under drought stress in the field at three U.S. locations over four years. Using soybean lines that had significant variation in canopy wilting, scientists identified genetic markers that were associated with delayed canopy wilting and increased agronomic performance. These genetic markers can be used to select and develop new drought-tolerant soybean varieties without the need for planting and evaluation in the field.


Review Publications
Menke, E., Steketee, C.J., Song, Q., Schapaugh, W.T., Carter Jr, T.E., Fallen, B.D., Zenglu, L. 2024. Genetic mapping reveals the complex genetic architecture controlling slow canopy wilting in soybean. Theoretical and Applied Genetics. 137(5). Article e107. https://doi.org/10.1007/s00122-024-04609-w.
Clevinger, E., Biyashev, R., Haak, D., Song, Q., Pilot, G., Saghai Maroof, M. 2023. Identification of quantitative trait loci controlling soybean seed protein and oil content. PLOS ONE. https://doi.org/10.1371/journal.pone.0286329.
Usovsky, M., Gamage, V.A., Meinhardt, C.G., Dietz, N., Triller, M., Basnet, P., Gillman, J.D., Bilyeu, K.D., Song, Q., Dhital, B., Nguyen, A., Mitchum, M.G., Scaboo, A. 2023. Loss-of-function of an a-SNAP gene confers resistance to soybean cyst nematode. Nature Communications. 14. Article e7629. https://doi.org/10.1038/s41467-023-43295-y.
Mahmood, A., Bilyeu, K.D., Skrabisova, M., Biova, J., De Meyer, E., Meinhardt, C., Usovsky, M., Song, Q., Lorenz, A., Mitchum, M., Shannon, G., Scaboo, A. 2023. Cataloging SCN resistance loci in North American public soybean breeding programs. Frontiers in Plant Science. 14. Article e1270546. https://doi.org/10.3389/fpls.2023.1270546.
Islam, S.M., Ghimire, A., Lay, L., Khan, W., Lee, J., Song, Q., Jo, H., Kim, Y. 2024. Identification of quantitative trait loci controlling root morphological traits in an interspecific soybean population using 2D imagery data. International Journal of Molecular Sciences. 25(9). Article e4687. https://doi.org/10.3390/ijms25094687.
Vuong, T.D., Florez-Palacios, L., Mozzoni, L., Clubb, M., Quigley, C.V., Song, Q., Kamad, S., Yuan, A., Chan, T., Mian, R.M., Nguyen, H. 2023. Genomic analysis and characterization of new loci associated with seed protein and oil content in soybeans. The Plant Genome. (16)4. Article e20400. https://doi.org/10.1002/tpg2.20400.
Miller, M., Song, Q., Li, Z. 2023. Genomic selection of soybean (Glycine max) for genomic improvement of yield and seed composition in a breeding context. The Plant Genome. 16(4). Article e20384. https://doi.org/10.1002/tpg2.20384.
Shea, Z., Singer, W., Rsso, L., Song, Q., Zhang, B. 2023. Determining genetic markers and seed characteristics related to high test weight in Glycine max. Plants. 12(16). Article e2997. https://doi.org/10.3390/plants12162997.
Araya, S., Elia, P.E., Quigley, C.V., Song, Q. 2023. Genetic variation and genetic complexity of nodule occupancy in soybean inoculated with USDA110 and USDA123 rhizobium strains. BMC Genomics. 24. Article e520 (2023). https://doi.org/10.1186/s12864-023-09627-4.
Yang, Q., Zhang, J., Shi, X., Chen, L., Qin, J., Zhang, M., Yang, C., Song, Q., Yan, L. 2023. Development of SNP marker panels for genotyping by target sequencing (GBTS) and its application in soybean. Molecular Breeding. 43. Article e26. https://doi.org/10.1007/s11032-023-01372-6.
Ma, G., Talukder, M.I., Song, Q., Li, X., Qi, L. 2023. Whole genome sequencing enables the molecular dissection and candidate gene identification of the rust resistance gene R12 in sunflower (Helianthus annuus L.). Journal of Theoretical and Applied Genetics. 136. Article 143. https://doi.org/10.1007/s00122-023-04389-9.
Soler-Garzon, A., Goldoff, D., Thornton, A., Swisher Grimm, K.D., Hart, J.P., Song, Q., Strausbaugh, C.A., Miklas, P.N. 2023. A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.). Frontiers in Plant Science. 14. Article 1215950. https://doi.org/10.3389/fpls.2023.1215950.