Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2023 Annual Report
Objectives
Objective 1: Efficiently and effectively acquire soybean genetic resources, maintain their safety, genetic integrity, health and viability, and distribute them and associated information worldwide.
Objective 2: Develop more effective germplasm maintenance, evaluation, and characterization methods, and apply them to priority soybean genetic resources. Record and disseminate evaluation and characterization data via GRIN-Global and other data sources.
Sub-objective 2a. Evaluate annual accessions for basic agronomic, descriptive and seed composition traits.
Sub-objective 2b. Conserve, regenerate, and distribute genetic resources and associated information.
Objective 3: Develop improved germplasm with increased yield by utilizing exotic soybean (Glycine max), wild soybean (Glycine soja) and wild perennial Glycine species; identify important introgressed genomic regions; and determine the impact of the introgressions.
Sub-objective 3a. Develop improved germplasm with increased yield using exotic and wild soybean, and identify integrated exotic DNA.
Sub-objective 3b. Identify important introgressed genomic regions associated with yield.
Sub-objective 3c. Understand the causes of genetic instability as seen in some mutants, as well as in some G. tomentella-derived lines that reverted from 2n=42 to 2n=40 chromosomes, that produce diversity in qualitative and quantitative traits.
Objective 4: Use appropriate genomic methods, including mapping and gene expression data, to identify genetic regions conferring quantitative defense to soybean pathogens and pests, discover useful genes, and work with breeders to deploy them in suitable germplasm.
Sub-objective 4a. Use GWAS, whole genome sequence assembly, and RNA-Seq to identify candidate defense-associated loci and genes to enhance resistance to S. sclerotiorum, rust, and the red-banded stink bug.
Sub-objective 4b. Verify candidate gene functions and usefulness of molecular markers related to defense-associated loci.
Approach
We will continue to expand the holdings of the USDA Soybean Germplasm Collection and optimize maintenance procedures. We will collect data on descriptive and agronomic traits, including photographs, and submit to GRIN-Global to facilitate the use of the Collection. High quality seeds will be maintained and distributed. We will use available breeding, genetic, and genomic tools to exploit the diversity of the Collection to increase seed yield and improved disease or pest resistance. Exotic accessions not in the commercially used gene pool will be used to develop high yielding experimental lines and populations to expand the genetic base of soybean production in the U.S. and identify new alleles from exotic germplasm that increase seed yield. Select lines derived from wide-crosses will be sequence analyzed to determine if genomic sections of the wild relative have been introgressed into the G. max genome. Genome-wide association mapping and analysis of gene expression data will assist in identification of candidate defense-associated genes, and the genes will be isolated for functional study.
Progress Report
This is the final report for this project which terminated in February 2023. See the report for the replacement project, 5012-21000-033-000D, “Maintenance, Characterization, Evaluation, and Enhancement of Genetic Resources in the National Soybean Germplasm Collection” for additional information. We summarize the progress toward each objective below for both this year and the entire project.
For Objective 1, over the life of this project, approximately 12,000 accessions have been grown for seed replenishment purposes, 7,480 germination tests have been conducted, and 151 germplasm releases and private cultivars have been added to the actively distributed soybean collection. Nearly 100,000 seed packets, encompassing 99% of the individual germplasm accessions were distributed in response to 2,100 seed requests.
In the past year, approximately 2300 new seed lots have been processed and added to the inventory for seed replenishment and distribution. Germination tests were conducted on 2,555 germplasm accessions with an average germination rate of 77%. Germination rates near or below 50% are regrown before being placed in active or long-term backup storage.
We activated 59 new accessions which included 12 germplasm releases, two public cultivars, and 45 private varieties with expired plant variety protection certificates.
In Urbana, Illinois, and Stoneville, Mississippi, a combined 1,740 accessions were field grown for observation and seed replenishment. In the greenhouse, 118 perennial Glycine and 169 wild Glycine accessions were also grown for observation and seed replenishment.
Different soybean accessions containing sterility and chlorophyll deficient traits have been verified throughout the life of this project plan as well as during the 2023 growing season.
For Objective 2, over the life of the project, many operational improvements have been made to the collection. These include fungicide treatment, prior to planting, of germplasm seed replenishment plots, an optical color sorter, which has reduced seed cleaning time by 50%, and greenhouse methods for growing difficult to cultivate Glycine species.
All 118 perennial accessions grown for seed increase have been tissue sampled and DNA isolated for genotyping in collaboration with Cornell University (Agreement #5012-21000-033-003S). This collaboration seeks to genotype various perennial Glycine to determine taxonomic differences at the genetic level.
An additional 3,000 images were captured this past year of the germplasm accessions. For the life of the project, more than 12,000 digital images have been taken in the field (whole plant) and lab (seed) with the majority being uploaded to GRIN, with the remainder to be uploaded in the calendar year.
For Objective 3, a large effort continues toward the genetic enhancement of soybean lines by combining the highest performing selection lines from the ARS Urbana, Illinois, soybean breeding program and recombining them with each other, and with sparingly or not previously used soybean germplasm accessions, to continue to broaden the overall performance of genetic diverse breeding material available to public and industry commercial soybean breeding programs. The contribution of soybean plant introductions on a pedigree basis to these lines is greater than 30%. In the Fall of 2022, 2,400 rows representing 200 unique breeding populations were used to harvest and individually thresh 3,300 plants. For yield testing, 2,619 single row, non-replicated plots, and 2,251 four-row, replicated plots were harvested. After selection for yield, lack of lodging, and protein and oil concentration, 2,300 segregating rows (from which individual plants will be selected), 3,300 single row, non-replicated plots, and 2,900 four-row, replicated plots were selected for planting in Spring 2023.
A total of 11 soybean lines from the Urbana, Illinois, breeding program were advanced to the USDA Uniform Soybean Tests, Northern Region, in addition to three lines advancing from year 1 to year 2 of the USDA Uniform Soybean Tests. We are also additionally participating in regional tests by growing the maturity group three and four field tests in the 2023 growing season. These tests represent the most advanced, elite soybean breeding lines from public breeding programs (universities and USDA) and provide the data necessary (10 or more locations spanning multiple states) to register soybean lines as commercial cultivars and to publish germplasm releases.
Over the course of this 5-year project plan, approximately 30,000 single and four-row plots have been planted, evaluated, harvested, and analyzed by the USDA-ARS soybean breeding program in Urbana, Illinois. Greater than 50 unique soybean lines have been evaluated in the USDA Uniform tests during that time, as well as more than 50 material transfer agreements have been granted for public and industry research programs to evaluate and breed with the soybean selections from this program.
For Objectives 3a and 3b, we have been focused on determining if soybean lines derived from crosses of G. max and G. tomentella contain any G. tomentella (PI441001) DNA. Sequence analysis was completed for 20 putative derived lines that were selected based on phenotypes that differed from the G. max parent Dwight (such as seed color or improved yield) as these lines were believed to be the most likely to have G. tomentella DNA introgression into their genomes. Aligning the shotgun genome sequences to high quality parental (Dwight and PI441001) genomes showed no discernible tomentella introgression into any of the lines. DNA samples from these 20 lines were also sent to an ARS lab in Beltsville for SNP analysis using the Illumina SoySNP50K genotyping arrays. Analysis of these samples showed that the regions that match DNA polymorphisms in PI441001, also mostly matched the DNA sequence of PI417089, a genotype that was discovered to have contaminated this project over a decade ago and whose DNA might have been integrated into some of the putative G. max by G. tomentella derived lines. The two Dwight x PI441001 lines that showed the most PI441001/PI417089 polymorphisms and phenotypic traits, will be whole-genome sequenced in the summer of 2023 to determine if the polymorphisms of these lines came from G. tomentella (PI441001) or the G. max contaminant PI417089.
For Objective 3c, we sent DNA from single plants and their offspring of the unstable Macon mutants. These lines were showing instability for traits such as flower color, pubescence color, and maturity, spanning 4 years of observations, to an ARS lab in Beltsville for SNP analysis using the Illumina SoySNP50K genotyping arrays. The genotyping analyses were consistent with an outcrossing event having occurred several generations earlier, and the ‘unstable’ polymorphisms are explained by the continued segregation of heterozygous traits because of the outcrossing.
For Objective 4a, we continue our efforts to characterize an allele of the Rpp1 (Rpp1b) locus that suppresses resistance to soybean rust and to verify the true Rpp1b gene. Transgenic plants expressing each of three different candidate Rpp1b genes were assayed for resistance to soybean rust, but their results were inconclusive, with none showing obvious resistance. We re-evaluated RNA-Seq data on soybean resistance response to the red-banded stinkbug and are preparing a manuscript describing the top 40 differentially expressed genes that responded to insect feeding.
For Objective 4b, we have been focused on developing markers that could differentiate Rpp1, Rpp1b and susceptible soybean genotypes from each other. Rpp1 and Rpp1b sequences were aligned and screened for unique restriction sites. Thirteen primer sets have been developed and tested on DNA from 16 plants of known rust resistance phenotypes. Only one primer set gave results consistent with the observed disease-reaction phenotypic data, suggesting genotypes may be more complex than sequencing data has shown so far. Large fragment cloning and sequencing of candidate gene regions of several lines is currently underway to help refine our approaches.
Accomplishments
1. Facilitated public, industry, domestic and international research with the USDA Soybean Germplasm Collection by distributing 19,163 seed packets for 11,679 unique accessions to fulfill 434 requests. Seed requestors are diverse in research goals and include applied plant breeding and screening for disease and pest resistance as well as basic research including genome sequencing, genetic mapping, and taxonomy studies. Within the past year, the ARS researchers in Urbana, Illinois, and the USDA Soybean Germplasm collection have been acknowledged in 216 journal articles. Specific outcomes include further dissection of resistance to soybean mosaic virus, Phomopsis seed decay, soybean rust, and soybean cyst nematode, as well as relating genomic regions to environmental adaptation of Chinese “land race” accessions and modern accessions. These examples of applied and basic research will lead to improved breeding outcomes against disease resistance and will lead to the genetic dissection of major climate adaption genes which will be crucial to the informed use of the more than 20,000 accessions available in the collection.
Review Publications
Zhang, Y., Blahut-Beatty, L., Zheng, S., Clough, S.J., Simmonds, D.H. 2022. The role of a soybean 14-3-3 gene (Glyma05g29080) on white mold resistance and nodulation investigations using CRISPR-Cas9 editing and RNA silencing. Molecular Plant-Microbe Interactions. 36(3):159-164. https://doi.org/10.1094/MPMI-07-22-0157-R.
Wei, W., Wu, X., Blahut-Beatty, L., Simmonds, D.H., Clough, S.J. 2022. Transcriptome profiling reveals molecular players in early soybean - Sclerotinia sclerotiorum interaction. Phytopathology. 112(8):1739-1752. https://doi.org/10.1094/PHYTO-08-21-0329-R.
Wei, W., Wu, X., Garcia, A., McCoppin, N.K., Gomes Viana, J.P., Murad, P.S., Walker, D.R., Hartman, G.L., Domier, L.L., Hudson, M.E., Clough, S.J. 2022. An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean. The Plant Journal. 113(5):915-933. https://doi.org/10.1111/tpj.16038.
