Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2022 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
For Objective 1, we sent backup seeds of 391 accessions to the National Laboratory for Genetic Resources Preservation. Approximately 2300 new seed lots have been processed and added to the active inventory for distribution. A large backlog of 3000+ seed lots to process still exists, primarily caused from vacancies due to retirements or resignations. Germination tests on 1,787 germplasm accessions identified 132 (7%) accessions with less than 50% germination. These accessions will be grown immediately in the next growing season to increase the initial quality of the seed lot before being placed in storage.
In Urbana, Illinois, and Stoneville, Mississippi combined, 1,800 accessions were field grown for observation and seed replenishment. In the green house, 80 perennial Glycine and 70 wild Glycine accessions were also grown for observation and seed replenishment.
Soybean accessions containing sterility and chlorophyll deficient traits are being verified in the 2022 growing season.
New soybean accession added to the collection included 69 expired patent lines, and 15 germplasm releases.
For Objective 2, fungicide seed treatment was used on all germplasm seed increase plots this season in Urbana, Illinois. Last year, the seed treatment pilot study showed that out of 100 treated split-plots, 75% had an increase in plant stand of 10% or more. This season, out of 885 seed increase plots, only 5 (0.56%) were rated as low stand (40 or fewer plants out of a 120-plant target stand). This compares to a past average of 4% or more low- stand plots per growing season.
Genotype-by-sequencing (GBS) analysis is being conducted on a portion of the perennial Glycine collection through collaboration with Cornell University. The primary goal is to clarify taxonomic classification and to separate complex species with multiple genomes, such as Glycine tomentella accessions, by which genome types they possess. This year, 74 accessions were tissue sampled in the greenhouse and DNA was extracted. These samples will be sequenced in the coming months with analysis to be completed upon completion of sequencing.
This past year, five soybean accession seed lots were discovered to contain one or more seeds of genetically engineered (GE) traits. These accessions were grown in the field, and single-plant, or bulk tissue sampled and tested again for GE contamination. Bulks and single plants which tested negative for GE contamination were harvested, processed, and further clean-seed grow-outs for replenishment will be conducted.
Digital images were taken of flowers, leaves, and midseason growth plots in Urbana, Illinois, and digital images of midseason growth plots of soybean and wild soybean were taken in Stoneville, Mississippi for a total of 3,500 total images. Approximately half of these images have been uploaded to GRIN with the remainder to be uploaded before the end of September 2022.
For Objective 3, a large effort continues to be maintained for the genetic enhancement of soybean lines by combining the highest performing selection lines from the USDA Urbana, Illinois, soybean breeding program and recombining them with each other, and with sparingly or never before 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. Across the entire breeding pipeline, the contribution of soybean plant introductions on a pedigree basis is greater than 30%. To that end, in the Fall of 2021, 2,000 rows representing 200 unique breeding populations were used to harvest and individually thresh 2,600 plants. For yield testing, 3,000 single row, non-replicated plots, and 3,800 four-row, replicated plots were harvested. After selection for yield, lack of lodging, and protein and oil concentration, 2,000 plant-pull rows, 2,600 single row, non-replicated plots, and 2,200 four-row, replicated plots were planted in Spring 2022. A decrease in single row and four-row testing effort is a combination of a concerted effort to downsize in order to better address the maintenance and regeneration of the soybean germplasm collection, but also reflects the resignation of a field technician in January of 2022 which remains vacant but is actively being attempted to be filled.
As a result of testing and selections made in the Fall and Winter of 2021, 44 soybean lines from the Urbana, Illinois breeding program were advanced to regional, cooperative testing, including eight lines which were entered into the USDA Uniform Soybean Tests, Northern Region. 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.
Efforts were continued to determine if any G. tomentella combined into putative G. tomentella x G. max derived lines. Now that we have full genomes of the parents (G. tomentella and G. max cv. Dwight), we re-evaluated data of short sequences and could not find any recombination based on that data. However, since the sequence data is sparse, and therefore not a complete representation of the genome, we sequenced 20 additional lines believed to be derived from G. tomentella crosses (good candidates based on phenotypes) at 30X coverage with a genotyping- by-sequencing method. This more complete sequence information has been aligned to the G. max Dwight and G. tomentella genomes and, although not 100% completed yet, we again have yet to find convincing introgression of G. tomentella DNA in any of the derived lines.
We have continued the genome annotation of G. tomentella PI441001. We have identified which chromosomes belong to which sub genome, and we have been working on the bioinformatics pipeline for gene calling using a combination of RNA sequences (we extracted and sequenced RNA from eight different tissues of the same PI441001 plant used for the genome sequencing) and the G. max reference genome.
For the unstable Macon cultivar mutants, we have DNA from four consecutive years of self-fertilized, unstable plants. DNA from these plants has been sent for genotyping to verify that the unstable phenotypes are not due to outcrossing. Once we have that genotype information, we will plant seed from select lines and grow plants for additional genome analyses (RNA-Seq, whole genome, DNA methylation) as needed.
For Objective 4, we now have transgenic seed from plants transformed with Rpp1b (gene which confers resistance to soybean rust) candidates as well as a putative suppressor of Rpp1/Rpp1b. Seed were grown, and plants screened for resistance to soybean rust. Results were not conclusive, and plants were assayed for the number of copies of the transgene. A private company determined copy number in these transgenic plants, and their results agreed 100% with our analysis of transgenic versus null plants. Knowing the copy number of the transgene will assist in selecting homozygous transgenic plants for assaying whole plants that are homozygous in the next generation. These plants are in the greenhouse maturing. Homozygous plants of the next generation will also be used for crosses to verify that we have identified the actual suppressor of Rpp1/Rpp1b. We have also re-evaluated our RNA- Seq data from soybean pods attacked by stink bugs using the latest genome assemblies. We have genome sequence assemblies for the resistant and susceptible lines of the stink bug studies and we are using these genomes to help identify function and location of differentially expressed genes.
Accomplishments
1. Facilitated public, industry, domestic and international research with the USDA Soybean Germplasm Collection. ARS researchers in Urbana, Illinois, distributed 21,985 seed packets involving 14,204 unique accessions through 358 requests, (including 26 international requests) to remain one of the only germplasm collections nationwide to distribute roughly as many seed packets as accessions maintained. 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. They rely on the availability of seed and information (nearly one million data points representing 180 traits) which ARS researchers in Urbana, Illinois, diligently maintain and develop. Impacts of such research on a global commodity such as soybean will include improvements in yield, nutrition (protein, oil, amino acids, etc) resiliency to climate change, emerging pests and diseases and other research objectives.
