Project : USDA ARS

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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

2025 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, we analyzed sequence reads from 63 common bean accessions, including 11 Meso accessions and 52 Andean accessions selected from different clusters of the Andean Bean Diverse Panel. The reads were aligned to the common bean whole genome sequence assembly Pvulgaris_442_v2.0.fa (v2). A total of 6,087,916 single nucleotide variants and 1,202,776 insertion/deletion sites were identified. After further elimination of tri-allelic variants, variants with missing sites greater than 50%, and variants with minor allele frequency less than 0.05, a total of 4,268,977 variants remained. Primer sets were designed for each variant based on 200 bp of sequence flanking the variant using primer 3. We further screened primer sets containing nonspecific sequences in the genome using e-PCR software and a total of 3,056,019 KASP markers were chosen. We randomly tested 50 primer sets for eight genotypes: Red Hawk, Lark, T-39, Laker, Stampede, Matterhorn, Bat93, and Jalo EEP558, and 96% of the primer sets successfully generated successful variant allele calls. The KASP markers are used in the laboratory and provided to collaborators at the USDA-ARS for fine mapping of genes controlling resistance to rust and anthracnose diseases. These markers will be a valuable resource for the community to identify genes controlling different traits and perform marker-assisted selection. Genomic loci controlling seed quality traits, including protein content, oil content, total protein and oil content, methionine, cysteine, lysine, and threonine content, were mapped in a nested association mapping population. The population consisted of 10 recombinant inbred line (RIL) families derived from 10 different wild soybean germplasms and crossed with a common cultivated soybean variety, “NC-Raleigh”. The parents and 1107 RILs had previously been grown for two years in Beltsville, Maryland, and Raleigh, North Carolina, and seed quality traits were measured. Analysis of the dataset showed that a higher number of recombination events were observed in the wild soybean progeny. Segregation distortion in almost all families was significantly biased toward alleles from the wild soybean parent. We also determined the effect range of QTLs controlling the protein, oil, and sulfur- containing amino acids (cysteine, methionine), lysine, and threonine content of wild soybean seeds, and detected new loci that showed large positive effects on cysteine, methionine from wild soybeans. This is the first study to reveal the genetic characteristics of wild soybean derived populations as well as the QTL landscape and influence magnitude of candidate genes from different wild soybean parents and controlling these traits. The results of this study were recently published in the peer-reviewed journal Theoretical and Applied Genetics. The information from this study provides new knowledge about wild soybean traits and will promote the use of wild soybeans to improve cultivated soybean seed composition traits. Based on this study, we further identified two major genomic regions that control high methionine and cysteine content in wild soybean, located on chromosomes 15 and 20. We further identified two F6 lines, C19-015 from PI 407020 x NC-Raleigh cross and C32-05 from PI 549032 x NC- Raleigh cross, which are heterozygous in the target regions of chromosomes 15 and 20, respectively. These two lines were planted in the field and seeds were harvested to produce segregating lines in these two regions. This population has recently been planted in the field and will be genotyped and phenotyped to locate genes controlling methionine and cysteine content. In addition, we genotyped more than 5,600 soybean and common bean germplasm and breeding lines developed by 13 researchers at universities or USDA-ARS research laboratories using the BARCBean12K assay developed in our laboratory for common beans and the BARCSoySNP6K, 3K, and SoySNP50K assays for soybeans. Through genotypic and phenotypic analyses, we mapped QTLs/genes controlling a variety of soybean traits, including root morphological traits, Fusarium graminearum infection, sucrose content, root rot, and seed iron and zinc accumulation, evaluated genetic diversity and seed composition stability in pan-African soybean varieties by collaborating with researchers at the University of Missouri in Columbia, Missouri; Virginia Tech in Blacksburg, Virginia; University of Georgia in Athens, Georgia; Virginia State University in Petersburg, Virginia; Purdue University in Lafayette, Indiana; and USDA-ARS in St. Louis, Missouri and Raleigh, North Carolina. In the common bean, we worked with researchers at the USDA-ARS in Prosser, Washington; Mayaguez, Puerto Rico; and Beltsville, Maryland, to develop SNP markers, fine-map the genes controlling resistance to rust and white mold and identify lines with broad genetic diversity and resilience to stress. These results were published in peer-reviewed journals. With funding from the National Science Foundation, we sequenced DNA from 440 edamame breeding lines at 30x sequence coverage per line and genotyped 430 edamame accessions using the SoySNP50K assay. DNA sequence analysis identified 9,943,310 genomic variants, including 174,892 variants that cause amino acid or protein variation and 118,386 variants that cause synonymous variation. These data will be shared with the PI at Virginia State University and will be used to examine genomic features and the molecular mechanisms that regulate seed filling and seed composition. We also randomly selected 50 soybean KASP markers from the soybean KASP marker dataset containing 1,494,086 markers and tested them on eight soybean germplasms (PI 549032, PI 407020, S-100, Richland, CNS, Hutcheson, Lee, and Williams 82). The results showed that 94% of the markers were successfully amplified and 72% of the markers were polymorphic. This dataset will be an excellent tool for locating genes controlling traits. Under Objective 2, our previous studies have shown that the breeding line VS12-0205 developed at Virginia State University completely restricted nodulation in the greenhouse after inoculation with a strain of rhizobia and growth in the field. The non-nodulation trait of VS12-0205 is controlled by a recessive gene and was determined to contain a novel gene/allele that controls non-nodulation. To locate and identify the gene in VS12-0205, we crossed Williams 82 (normal nodulation) with VS12-0205 (non-nodulation) and obtained a total of 3,000 F2 seeds. These seeds will be grown in the field to examine nodule segregation and determine the gene location in the genome. Under Objective 3, the Rhizobium Collection has been surveyed to determine which cultures need to be re- stocked, and which cultures are over 30 years old and need to be replaced. The online database has been updated to include newly acquired cultures, and information concerning the geographic origin of each accession. Efforts to back up the Collection at the long-term storage facility at Fort Collins, Colorado, continue, with the assembly and quality control of 288 of R. leguminosarum phaseoli) and 107 Bradyrhizobium spp for long term backup. All stakeholder requests were met in a timely manner. Over 120 strains were provided to state, private universities and Federal Research Institutes. Rhizobium cultures and technological support were also provided to U.S. private commercial enterprises specializing in the production of rhizobium inoculants for legume crops, including Bio-Next Inc., Wichita, Kansas, and Vison Biologics, in Henrietta, Texas. Over 500 accessions that needed replenishment or were over 30 years old have been re-stocked in the USDA-ARS Rhizobium Collection. Under Objective 4, DNA has been isolated from 54 Rhizobia strains from beans, soybeans, and other legumes, and an additional 200 strains are currently being recovered and activated for DNA extraction. DNA from these strains, as well as DNA from strains extracted in subsequent years, will be sequenced and analyzed in conjunction with sequence reads from previously sequenced strains to identify variation among strains. This information will be used to develop a core strain collection and high-throughput genotyping assays for Rhizobia strain identification. In collaboration with the Department of Energy's Joint Genome Institute in Berkeley, California, genome sequences of more than 400 Rhizobia strains have been assembled based on long sequence reads.

Accomplishments
1. Explore landscape rare-allele variants in cultivated and wild soybean genomes. Rare allele variants are alternative forms of a gene arising from a mutation that occur at low frequencies. These variants are important for crop improvement because some variants may be associated with important traits such as disease resistance, insect resistance, drought tolerance, and seed composition. However, the distribution of these variants throughout the DNA of chromosomes (genome) and the mutation effects on gene function and protein synthesis in soybeans have not been reported. USDA-ARS researchers at Beltsville, Maryland, and St. Louis, Missouri, and their collaborators analyzed the sequences of 1,556 cultivated and wild soybean genomes and used them to identify and functionally predict the biological functions of all variants, especially rare allelic variations in cultivated and wild soybeans. They concluded that domestication and breeding selection have greatly reduced the genetic diversity of cultivated soybean compared to wild soybean. They found that rare- allele variants may result in mutations with an impact on functions in 40-60% of genes. This is the first comprehensive study of rare allelic variation in wild and cultivated soybean genomes and its potential effects on gene function. This information is a source for future genetic and genomic research by public and private soybean breeders and geneticists, as these rare alleles may have untapped potential to benefit soybean growers by improving crop stress resistance, yield, and adaptability to a changing environment.

2. Tools to rapidly monitor and identify unique DNA sequences controlling import soybean traits. Methods for rapidly detecting and identifying unique DNA sequences (markers) associated with production traits are crucial for determining which chromosome regions contain genes controlling traits, enabling breeding selection, and predicting trait performance. The two most used assays, SoySNP50K and BARSoySNP6K, have 50,000 and 6,000 DNA markers respectively. Despite the success of the common assays, both platforms are very costly and have more information than needed by soybean breeders to predict breeding value of offspring, select hybrid parent (genetically distinct traits) and to use for the early identification of progeny across thousands of breeding lines. Breeders need a cheaper, less rigorous and more rapid platform with specific markers for economic traits such as seed composition and resistance to disease and pests. USDA-ARS scientists in Beltsville, Maryland, working with U.S. breeders, have developed two highly efficient marker assays with core sets of 3,000 and 1,000 markers, including trait- associated markers. These assays were commercialized by Illumina, Inc. and AgriPlex Genomics in Cleveland, Ohio, respectively, and are used by soybean researchers in the public and private sectors. The new assay will serve as an additional low-cost tool for genetic, genomic and breeding research and will address the soybean community's need for low marker density assays to achieve its research goals.

3. Identified loci controlling seed composition traits from wild soybean. Wild soybean is a valuable source for improving disease resistance, stress tolerance, seed protein content, and seed sulfur amino acid concentrations. Many past studies have focused on seed composition traits based on cultivated soybean populations, but wild soybean has been largely overlooked. In this study, USDA-ARS scientists in Beltsville, Maryland, and collaborators explored the genetic potential of wild soybean in improving seed composition traits in cultivated soybean. They developed a population of 10 wild soybean germplasm crossed with the cultivated variety. After identifying genetic differences linked with traits in more than 1,100 progeny and measuring seed composition of the progeny planted at two locations and over two years, the researchers revealed the genetic characteristics of wild soybean-derived populations, landscapes, and the extent of influence of genetic differences and candidate genes controlling traits from different wild soybean parents. They also identified several regions in chromosomes and candidate genes from wild soybean that could be used to improve the composition of cultivated soybean seeds (loci). This is the first report to show the genetic characteristics of wild soybean population and its potential to improve seed composition in cultivated varieties based on progeny from many wild soybean germplasms. The findings highlight the importance of using wild soybeans as a genetic resource to enhance seed composition traits in soybean breeder’s breeding programs.

U.S. DEPARTMENT OF AGRICULTURE

Soybean Genomics & Improvement Laboratory: Beltsville, MD