Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2019 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 (Efficiently and effectively acquire soybean genetic resources, maintain their safety, genetic integrity, health and viability, and distribute them and associated information worldwide):
10,869 seed lots from 6,420 accessions were distributed in response to 355 requests from 264 individuals. A total of 1,024 germplasm increases were grown and harvested, and 1,308 germplasm increases were planted for seed collection maintenance. A set of 1,025 newly increased seed inventories were cleaned and made available. We added 29 new germplasm releases, 1 isoline, and 3 cultivars to the collection. We increased seed of 94 new perennial Glycine accessions that were received in 2017. At the start of FY19, there were 150 Glycine perennial accessions (52 G. tomentella, 50 G. tabacina, and 48 accessions of unknown species) for which there was no information on chromosome numbers. During FY19, chromosomes were counted and photographically documented for 78 accessions.
We initiated a screen of the collection for the presence of the glyphosate-resistance trait. A total of 10,032 accessions were sampled, and another 2,800 accessions have been packaged for testing this fiscal year. Of these 10,032 accessions screened, 12 were positive and were removed from the active collection. In addition to these 12 positives, 24 accessions had very low levels of contamination. Seed distribution of these 24 accessions has been stopped until new seed can be harvested from individual plants that test negative. We also initiated germination testing of each accessions of the collection, and 400 samples have been subjected to germination testing.
For 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):
One of the goals of the USDA is to have the entire soybean germplasm collection genotyped with soybean 50,000 single nucleotide polymorphism (SNP) microarrays. After the initial efforts to genotype the entire collection, about 1,400 accessions remained that either did not get genotyped, or that needed repeating. In FY19 we have been finishing the DNA extractions from the remaining accessions, and all extractions will be sent to Beltsville for analysis on soybean 50,000 SNP microarrays by the end of FY19.
We uploaded to GRIN 2,274 accession records and 13 crop trait images, as well as 12,930 crop trait observations including 440 of soybean cyst nematode screened in Stoneville, Mississippi and 353 from plant variety protection certificates and crop registration descriptions.
We purchased and installed a dust collector for our thrashing room to minimize dust exposure to employees.
We planted nine field tests (5,440 four-row yield plots) at 2 locations, Urbana and Ivesdale, Illinois. In FY19, we harvested 3,834 four-row yield plots and 3,575 one-row plots, and seven large seed increases. Individual plants were pulled from 101 genetic populations (GPOPS) and 1,010 F2 populations grown in 2018. We planted 5,025 one-row yield plots in Urbana. There are 17 large seed increases for the Uniform and Preliminary tests, and 125 GPOPS and 526 F2 populations being grown in 2019. We sent seed from ARS breeding lines to eight collaborators in a project to identify elite soybean breeding lines that yield well and have seeds with above average protein content. Of the 249 breeding lines tested alongside high-yielding check varieties, 155 (62%) were selections from the Urbana ARS breeding program. The ARS lines all have some exotic ancestry from the USDA Soybean Germplasm Collection, and 34 of the lines originate from an interspecific cross with an accession of the perennial species Glycine tomentella. Twenty other lines have the wild annual soybean species G. soja in their ancestry.
For several years, we have been following plants from a mutagenesis study that have not been phenotypically stable. In the 2018 growing season, we identified two rows of plants that were highly variable (should have been highly homozygous and therefore highly homogeneous) from plant rows originating from each of the two original lines. We collected seed and leaf tissue (for DNA extraction) from all the plants of these four rows and planted those seed in 2019 to again look for rows that are highly variable. Rows have been identified in the 2019 growing season that are highly variable, and DNA is currently being extracted from these and their progenitor plants for genetic analyses.
In FY19 we continued to analyze the alignments of assembled genome sequences of two lines derived from crosses between soybean (G. max; 40 chromosomes) and one of its wild perennial relatives (G. tomentella; 78 chromosomes) and the parents used in the crossing, in an effort to identify possible regions of DNA exchange. We have identified many regions of difference scattered across the genomes suggestive of random differences (possibly mutagenesis as a result of transposon activation); however, we still cannot rule out introgression. We sent leaf tissue of G. tomentella to a company to assist in the genome assembly of this tetraploid, but it has been difficult to assemble the genome of G. tomentella, and the company asked for new tissue that was sent out July 15, 2019. The G. tomentella plant material was from an accession that is publicly available in the USDA Soybean Germplasm Repository.
For 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):
For a genome wide association study to identify possible soybean quantitative resistance to soybean rust, we used published phenotypic data on 3,215 accessions from the USDA Soybean Germplasm collection that already had been genotyped on soybean 50,000-SNP microarrays. The phenotypic and genotypic data were filtered to include only the most reliable data, and approximately 15,000 single nucleotide polymorphisms and 2,000 lines were kept. The analysis identified three significant loci as being associated with quantitative resistance.
For 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):
Working in collaboration with researchers at Agriculture and AgriFood Canada, a site-specific mutation was introduced into a 14-3-3 gene that we previously showed by RNA-mediated gene silencing to affect nodulation and defense to white mold caused by the fungus Sclerotinia sclerotiorum. In FY19 we evaluated the 14-3-3 mutant for ability to nodulate (conducted by the ARS scientists in Urbana, Illinois) and to defend against white mold (conducted by the Canadian group).
We continued our efforts to identify genomic regions of a dominant susceptible plant that can suppress the rust resistance gene Rpp1. The suppressor region was cloned out from the dominant susceptible as well as two resistant PIs for comparison. The clones were identified through fosmid library screening and sequenced. Candidate genes and possible small RNA targets were searched, and expression levels are being evaluated with gene-specific quantitative reverse transcription polymerase chain reaction. Genomic rearrangements were identified by aligning and comparing the sequences between the different genotypes. No obvious small RNA sequences have been identified, but a candidate dominant susceptible gene has been selected. To test the hypothesis that this candidate gene suppresses Rpp1 function, gene overexpression, virus-induced gene silencing, and stable soybean transformation are being pursued.
To establish soybean transformation, we have been following protocols for the cotyledonary node transformation method with Agrobacterium containing a binary vector with a beta-glucuronidase reporter gene, that turns the soybean tissue blue if transformed. A total of 10 experiments were conducted in FY19. One test simply looked for genotypes that had the highest rate of transformation based on color change. We also tested if adding amino acids to the culture media could improve transformation rates.
Accomplishments
1. Identified chromosomal loci associated with quantitative resistance to soybean rust. Soybean rust can cause significant damage to soybean crops grown in the southeastern United States. Populations of the fungus that cause soybean rust are very diverse and adapt quickly to overcome single dominant genes for rust resistance. Quantitative resistance, where multiple genes each contribute a small portion of disease resistance, has the potential to provide more durable resistance to the disease and reduce grower inputs by reducing reliance on synthetic fungicides for control of soybean rust. To identify regions of soybean chromosomes associated with quantitative resistance to soybean rust, ARS scientists in Urbana, Illinois, collaborated with University of Illinois researchers to conduct a genome wide association study of 3,215 accessions from the USDA Soybean Germplasm Collection and publicly available molecular marker data. The analysis identified three significant loci as being associated with quantitative resistance to soybean rust. The discovery and mapping of chromosomal regions containing quantitative genes for resistance to soybean rust could be combined in new cultivars to reduce fungicide use and to protect soybean yields in production areas of the United States where soybean rust is common.
Review Publications
Smith, J.R., Gillen, A.M., Nelson, R.L., Bruns, H.A., Mengistu, A., Li, S., Bellaloui, N. 2019. Registration of high-yielding exotically-derived soybean germplasm line LG03-4561-14. Journal of Plant Registrations. 13:237-244. https://doi.org/10.3198/jpr2018.09.0061crg.
Grando, C., Amon, N.D., Clough, S.J., Guo, N., Wei, W., Azevedo, P., Lopez-Uribe, M.M., Zucchi, M.I. 2018. Two colors, one species: The case of Melissodes nigroaenea (Apidae: Eucerini), an important pollinator of cotton fields in Brazil. Sociobiology. 64:645-653. https://doi.org/10.13102/sociobiology.v65i4.3464.
Ray, J., Yang, X., Kong, F., Guo, T., Deng, F., Clough, S.J., Ramonell, K. 2018. A novel receptor-like kinase involved in fungal pathogen defense in Arabidopsis thaliana. Journal of Phytopathology. 166:506-515. https://doi.org/10.1111/jph.12711.
