Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2021 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 78 accessions to the National Laboratory for Genetic Resources Preservation (NLGRP). Currently, 99% of the collection is backed up at NLGRP in Fort Collins, Colorado and 89% is backed up at the Svalbard Arctic Seed Vault.
Approximately 2400 new seed lots have been processed and added to the inventory for distribution. As new seed lots are added, packets of seed for germination tests have been set aside. A backlog of 3000+ accessions still needs to be processed, not including this year’s harvest. Lack of personnel is the primary reason for the backlog. A set of 500 accessions were transported to Ames, Iowa and cleaned with an optical color sorter. Percent germination for 793 samples was conducted. Average germination rate was 75% and 100 samples were below 50% germination rate and designated for replanting.
In Urbana and Stoneville combined, approximately 2,868 accessions were field grown for observation and increase. Additionally, 387 pots of 78 perennial accessions were planted, maintained, and harvested from the greenhouse throughout FY21.
Light-tawny pubescence soybean accessions are being verified this year.
The soybean crop vulnerability report was updated.
New soybean accessions added to the collection included 17 expired PVP lines and 2 germplasm releases.
For Objective 2, a fungicide seed treatment was tested on 100 planted accessions this year, with treated and untreated rows of the same accession compared. Nearly every plot had early growth improvements on the rows that were treated, with some plots ranging from nearly full stands (160 plans) on the treated rows compared to less than 10 plants on the non-treated rows of the same accession. A fungicide seed treatment will be applied to all future increase plots to improve harvested seed lots.
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, such as Glycine tomentella accessions by which genome types they possess. In FY21, 116 accessions were tissue sampled in the greenhouse and DNA was extracted.
We are also in the second consecutive year of sending one sample of every accession in the germplasm collection to the University of Nebraska for a genetic sequencing project. We distributed 10,000 for this project accessions for this project and all are anticipated to be sequenced in FY22.
We screened an additional 5,100 accessions for the presence of genetically engineered (GE) traits. Eight seed lots were found positive ranging from 1-10 plants out of 200 that were resistant to the herbicide. These accessions were planted this spring and will be tissue sampled and tested. Plants that test negative will be harvested and increased further to be maintained in the collection. Up to this point, every soybean accession in the germplasm collection has been confirmed to be free of transgenes but contamination via off-type plants, pollen introduction via insects, and donated germplasm having GE contamination upon receipt, has required this additional testing.
Staff of the germplasm collection are still prioritizing obtaining images of the accessions, including seed, green plants, flowers, and mature plants. To this end, 4,400 images were uploaded to the GRIN-Global public website.
For Objective 3, in the fall of 2020 seeds were harvested from 760 breeding lines grown in 22 replicated yield tests planted in Savoy, Illinois. A majority of the lines were derived from crosses with exotic germplasm accessions from the USDA Soybean Germplasm Collection. Approximately 15% of the Urbana breeding lines were derived from crosses with either wild soybean (Glycine soja) or the perennial Australian species G. tomentella.
The 2020 yield data was a total loss due to severe flooding soon after planting and hail damage in July 2020. Superior Urbana lines grown in cooperative tests planted in other locations and states were selected based on their performance across locations in 2020.
Extramural funding was received from the United Soybean Board for two projects that have the objective of increasing seed protein content without a significant yield reduction. The research approach for both is to introgress (i.e., breed in) novel genes from exotic soybean germplasm accessions to generate lines with a different gene pool than current commercially grown cultivars. Breeding lines derived from crosses with exotic germplasm accessions have shown the potential to produce progeny with both competitive yields and a higher percentage of protein. Up to 78% of the genes in some promising Urbana ARS lines from this project were inherited from exotic ancestors. Breeding crosses were also made with lines that inherited 13% of their DNA from wild soybean (G. soja) or approximately 6% of their genes (theoretically) from a Glycine tomentella ancestor.
Seed from 70 soybean germplasm accessions reported to have high seed protein and low seed oil were harvested in late 2020 and sent to an ARS collaborator in Raleigh, North Carolina for analysis of seed components. Hybrid seed were obtained from crosses made with 15 high-protein accessions that had few disease symptoms and agronomically desirable traits. Several high-yielding ARS lines from Urbana were also crossed with ‘Highpro1,’ an ARS line developed in Wooster, Ohio that carries a high protein gene originating from the Korean variety ‘Danbaekkong’ which has a less detrimental effect on yield.
Hybrid F1 seeds from each of 84 different crosses made during the summer of 2020 were sent to a winter nursery in Puerto Rico in December 2020. F2 seeds from the F1 plants were planted in rows in May 2021 to obtain F3 seeds.
In May 2021, 228 advanced soybean lines were planted in replicated yield tests. Single-row plots totaling 3,243 were planted in addition to hand planting 288 F1 plants.
In May 2021, the following research plots were grown for the Soybean Germplasm Collection Breeding program:
2,392 plant selection rows
6,313 single-row, single replicate early generation yield plots
4,028 four-row, two replicate yield plots
To be able to clearly determine introgression of G. tomentella DNA into the G. max genome of wide cross progeny, we need a high-quality genome assembly of G. tomentella which is a more difficult genome to assemble than G. max due to the multiple duplicated genomes. Therefore, we sequenced G. tomentella (PI441001) and Dwight (the G. max parent of these derived lines). In FY2021 we have received the genome sequences and have been analyzing them, in addition to analyzing published GBS sequence data from 78 derived lines. The genomes of both PI441001 and Dwight assembled very well, and we have complete contigs for all the 39 chromosomes of PI441001, and the 20 chromosomes in Dwight. Published RNA-Seq data has been retrieved of the proposed subgenomes of PI441001 and we are in the process of cleaning and aligning those reads to identify which chromosomes belong to which subgenome. We will also use the RNA-Seq data to help annotate the PI441001 transcripts. We are trying multiple conditions for alignment of the GBS reads to the new genomes of PI441001 and Dwight, in attempts to identify if any of the putative derived lines have G. tomentella DNA.
Since about 2005, we have been following plants from a mutagenesis study that have not been phenotypically stable. In the 2020 growing season, we identified five short rows of plants that were still highly variable (should have been highly homozygous and therefore highly homogeneous). In FY21 we collected seed and leaf tissue (for DNA extraction) from all the plants plus various control plants (100 total). DNA will be used to genotype the lines to determine if they resulted from outcrossing, or if they are highly homologous to the parent plant. SNP analyses will be conducted on the plants that are clearly derived by mutation to search for common mutation(s).
For Objective 4, we previously conducted short-read sequencing of three soybean genotypes. In FY21, our Brazilian collaborators have been annotating the genome assembly of two of the genomes—IAC-100 (resistance to stink bugs, rust and white mold) and CD-215 (susceptible) to stink bugs, rust and white mold). RNA-Seq expression data from these two genotypes is being used to identify intron/exons.
We continued our efforts to genetically characterize a dominant susceptible (DS) soybean that can suppress Rpp1-mediated rust-resistance in soybean. The suppressor region was cloned from the DS plant through fosmid library screening, as have the Rpp1 candidate genes. We used yeast 2-hybrid protein-interaction studies to show that DS most likely is functioning through direct binding of the DS protein to the Rpp1 protein, and inhibiting its ability to transduce an adequate defense response. Stable soybean transformants that contain the DS sequence as well as Rpp1 candidates are being produced through a transformation service. We also conducted a population level search of the DS allele in multiple soybean populations using published sequence data and found that is distributed across a diversity of soybean accessions, as well as in G. soja.
Accomplishments
1. The USDA Soybean Germplasm Collection distributed 24,930 seed packets for FY21. One of only a few germplasm collections to distribute a greater number of seed packets than the total number of accessions we curated. ARS researchers at Urbana, Illinois, distributed 24,930 seed lots from 13,582 accessions from the USDA Soybean Germplasm Collection in response to 426 requests from 269 individuals. There were 381 domestic requests (89% of the total) with a total of 22,870 seed packets sent to researchers from 36 states. There were 2060 seed packets of 45 orders sent to scientists in 17 countries. Sixteen requests were made for 266 seed packets of 209 perennial Glycine accessions. The number of requests and diversity of requestors (public, private, international) highlights the need for unique soybean germplasm to fulfill basic and applied research goals, and the ability of the USDA Soybean Germplasm Collection at Urbana, Illinois to meet this need.
2. Identified unique, high-yielding soybean breeding lines. Genetic diversity among modern soybean cultivars is very narrow due to a strong historical emphasis on yield, limiting the potential to improve soybean for traits like increased seed protein or resistance to pests and diseases. ARS researchers at Urbana, Illinois, developed soybean lines that yield equivalent to commercial checks, despite having either inherited a large percentage (>50%) of their genes from exotic germplasm accessions, or having wild soybean ancestors. In the 2020 USDA Northern Uniform Soybean Tests, ARS researchers at Urbana, Illinois, submitted multiple soybean lines that finished in the top 50% of all entries for yield. This performance has resulted in seed requests from 14 Midwestern universities and private companies to request seed for testing and genetic improvement research. Getting this unique, diverse material into research programs that produce commercial soybean varieties will help broaden the genetic base of soybean in the United States.
3. Identified competitive soybean lines with elevated protein content. In the commercially available genetic base of soybean, higher yields are associated with lower protein content, to the extent that protein content has slowly been reduced. Soybean processors need a soybean meal with at least 48% protein. This can usually be achieved with soybean seed protein contents above 35%. Utilization of unique genes from exotic germplasm can make it possible to increase protein content without a corresponding yield loss. ARS scientists at Urbana, Illinois, developed and identified soybean lines with elevated protein content of 35-37% (commercial average is 33-34%) and yields comparable to commercial cultivars, including some containing 50% or greater exotic soybean germplasm. These included several lines that had a Glycine soja (wild soybean) accession as one of the parents. This indicates a substantial yield component is potentially being brought from the Glycine soja parent. This demonstrates that yield and protein can be increased in parallel by incorporating unique genes from diverse accessions in the USDA Soybean Germplasm Collection. Increasing genetic diversity in U.S. soybean breeding programs will benefit producers and breeders by introducing unique genes to enhance yield, resistance to disease, and important seed quality traits.
