Location: Soybean/maize Germplasm, Pathology, and Genetics Research
2020 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
Objective 1. Efficiently and effectively acquire soybean genetic resources, maintain their safety, genetic integrity, health and viability, and distribute them and associated information worldwide.
We distributed 11,858 seed lots from 8,618 accessions from the USDA Soybean Germplasm Collection in response to 180 requests from 136 individuals. There were 160 domestic requests (89% of the total) with a total of 11,286 seed packets representing 8,394 accessions sent to 118 researchers from 35 states. There were 572 seed packets of 554 accessions in 320 orders sent to 18 scientists in 12 countries. Eight requests were made for 104 seed packets of 93 perennial Glycine accessions. We also sent backup seeds of 166 accessions to the National Laboratory for Genetic Resources Preservation (NLGRP). Currently, 99% of the collection is backed up at NLGRP and 89% is backed up at the Svalbard Arctic Seed Vault.
Approximately 2000 new seed lots have been processed and added to the inventory for distribution. As new seed lots are added, over 1000 packets of seed for germination tests have been set aside. Percent germination for 501 samples was conducted in FY20.
Approximately 2800 seed lots were harvested in Urbana and Stoneville in the fall of 2019 or in Costa Rica in the spring of 2020. We planted 2200 accessions for seed increases in FY20.
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 Germplasm Resorces Information Network (GRIN)-Global and other data sources.
We determined chromosome number for 88 Glycine tomentella in the collection with previously unknown chromosome numbers.
Genotpe-by-Seqencing analysis on some of the perennial Glycine accessions is being done through collaborators at Cornell University to help clarify difficult taxonomic classifications. In FY20, we extracted DNA from 66 for this analysis. Additionally, we have begun screening the entire collection with a different set of 50K SNPs through collaboration with the University of Nebraska. The first set of 2000 accessions were sent to him in October 2020.
It has recently come to our attention that the germplasm accessions need to be screened for possible contamination from transgenic soybean. We screened 1,195 accessions in FY20 for the adventitious presence of the genetically engineered trait Roundup Ready (RR) and found five seed lots were RR+. Seed distribution of these has been stopped until new seed (sown in short rows in May 2020) can be harvested from individual plants that test negative. Plants from these short rows (50 or fewer plants) were just sampled and all were negative, so we will be able to use seed from plants for future distribution. One bulk sample of 20 seed lots tested positive and will need further testing. We uploaded 6300 images to GRIN of 3000 accessions.
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.
In the fall of 2019 soybean were harvested from field plots in three locations. All lines were derived from crosses with at least one exotic plant introduction, including Glycine soja or Glycine tomentella accessions. In 2019, 1,542 lines were evaluated in yield tests and 4,625 plant rows were grown. Plant selections were made from 125 F5 populations and 52 F2 populations. F1 seed were obtained from 35 different crosses. Evaluation MTAs for Urbana ARS lines were executed with six university and two private industry breeding programs. Breeding MTAs for Urbana breeding lines were executed with four universities and two companies.
Urbana ARS lines were evaluated in the 2019 USDA Northern Preliminary Test (PT) IIA (four lines), PT IIIA (eight lines), PT IV (five lines) and Uniform Test IV (five lines). The yields for maturity group (MG) II lines equaled or exceeded those of three of the four checks in the PT IIA test. In the PT IIIA test, three Urbana lines had yields equivalent to the top line among the 29 entries, ranking 3rd, 4th and 7th, respectively. LG17-8888 had the highest yield of the 24 entries in PT IV, and LG17-8885 ranked third in that test. In the UT IV advanced test of the best MG IV lines from 2018, LG16-4655 had the highest yield, LG16-4642 ranked 2nd, and LG16-4634 and LG16-4644 ranked 4th and 7th, respectively.
Urbana ARS lines performed very well in United Soybean Board-funded multi-state tests to evaluate yield and seed composition traits in 2019. LG16-21134, from an interspecific cross with Glycine tomentella, was the highest-yielding line in the 2019 advanced MG II test. In the preliminary MG II test, LG18-1148 was 3rd among the 36 entries, and other Urbana lines ranked 6th and 7th. All three lines had 11 different “exotic” progenitors. LG18-1148 and LG18-1152 inherited 37% of their genomes from exotic sources, and LG18-1091 76%. In a test of advanced MG III lines, LG17-7150 had the third highest yield and 23 other Urbana ARS lines had yields that were numerically higher than one check variety. Although LG17-7150 carries only about 7% exotic genes, some are from wild soybean (Glycine soja) and from Glycine tomentella. In a preliminary test of 32 MG III lines, LG18-2636 and LG18-2627 (both with 16% exotic DNA) had the highest and third highest yields.
To be able to clearly determine introgression of Glycine tomentella DNA into the Glycine max genome of wide cross progeny, we need a high-quality genome assembly of Glycine tomentella which is a more difficult genome to assemble than Glycine max due to the multiple duplicated genomes. We sent tissue to a service provider for sequencing.
Since about 2005, we have been following plants from a mutagenesis study that have not been phenotypically stable. In the 2019 growing season, we identified four short rows of plants that were still highly variable (should have been highly homozygous and therefore highly homogeneous). We collected seed and leaf tissue (for DNA extraction) from all the plants (51 total) of these four rows and planted those seed in 2020 to again look for rows that continue to be unstable.
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.
We previously conducted Illumina and 10X Genomic’s based sequencing of three soybean genotypes. In FY20, our Brazilian collaborators have been refining the genome assembling 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).
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. In FY20 we verified that the 14-3-3 gene is required for maximal nodulation (conducted by the ARS scientists in Urbana, IL) and that it is involved in defense to white mold caused by the fungus Sclerotinia sclerotiorum (conducted by the Canadian group).
In FY20 we continued to conduct various Genome-wide association study (GWAS) analyses using different phenotypic and genotypic data filtering approaches and statistical analyses of previously published defense responses to soybean rust and concluded that at least six significant loci are significant. The two most significant regions (on chromosomes 18 and 6) overlapped with the previously mapped rust resistance loci Rpp1 and Rpp3. The other significant regions have not been reported previously.
We continued our efforts to identify genomic regions of a dominant susceptible (DS) soybean that can suppress Rpp1-mediated resistance to soybean. The suppressor region was cloned out from the DS plant through fosmid library screening. To test the hypothesis that this candidate gene suppresses Rpp1 function, virus-induced gene silencing (VIGS) experiments were conducted in FY20 using various genetic backgrounds including F1 progeny of selected crosses. The VIGS experiments have been giving results supporting that we have identified an Rpp1 suppressing region, with the best VIGS construct imposing a two-fold reduction in spore production and in expression of the DS candidate gene. We also initiated the generation of stable soybean transformants to contain the DS sequence through the University of Wisconsin.
To help identify how the Rpp1 suppression sequence was generated, fosmid libraries were constructed in FY20 from three of the parents used to create the DS line. Once the Rpp1 region in these cultivars are assembled, they will be compared to the Rpp1 region in the DS to see how the sequences changed during breeding.
Accomplishments
1. Urbana ARS breeding lines with 25 to 62% exotic DNA were among the highest yielding maturity group IV lines in multistate cooperative evaluations in 2019. Genetic diversity among modern soybean cultivars is very limited and restricts the potential to increase yields and improve resistance to biotic and abiotic stresses. ARS researchers in Urbana, Illinois, developed genetically diverse breeding lines by crossing elite, high-yielding lines with plant introductions (PIs) from Asia. LG15-4655, LG15-4642, LG15-4634 and LG15-4644 were the highest yielding, and second, fourth and seventh highest yielding lines out of 19 entries in the 2019 Northern Maturity Group IV Uniform Test, respectively, with yields equal to or better than the best check line. Based on their pedigrees, each of these lines inherited 25% of its genes from the Chinese germplasm accessions PI 561319A and PI 574477. Line LG15-4348, with 62% exotic DNA, ranked ninth for yield in the test, but its yield was statistically equivalent to two of the three check lines. It has a unique pedigree and is derived from crosses with six soybean germplasm accessions and one accession of wild soybean (Glycine soja). All of these lines also had high yields in 2018. These results demonstrate the potential to increase genetic diversity in high-yielding soybean lines by combining genes from local elite parents and exotic germplasm, including wild and perennial Glycine accessions.
2. Breeding lines with exotic pedigrees and high yield met or exceeded a target of 48% meal protein and 11 pounds of oil per bushel in 2019. North American soybean processors desire soybean seeds with at least 47.5% meal protein and 10 pounds of oil per bushel. Due to a negative correlation between seed protein and seed yield and oil content in modern cultivars, increasing protein levels in cultivars derived from elite x elite crosses is seldom successful. In a collaborative project to circumvent this obstacle, ARS scientists in Urbana, Illinois, developed and selected breeding lines with at least 48% meal protein and 11 pounds of oil per bushel. In multistate evaluations of advanced breeding lines in 2019, four of the seven highest yielding entries in the maturity group (MG) III test (41 entries total) and six of the seven highest yielding MG IV lines out of 16 entries were Urbana ARS lines. These lines had also performed well in 2018. In preliminary multistate tests, four of the six highest yielding lines in a MG III test with 33 lines were Urbana ARS lines and eight ARS lines in a MG IV test out-yielded two of the three check lines, while still meeting the minimum protein and oil content criteria. The percentage of exotic germplasm accession DNA in these lines ranged from 3 to 65%, and about one-third of them had wild annual or perennial Glycine progenitors in their pedigrees. These results illustrate the potential to increase soybean seed protein without a concomitant decrease in yield by utilizing genes from accessions in the USDA Soybean Germplasm Collection that are absent from the narrow germplasm pool of modern North American cultivars.
Review Publications
Valquíria Dos Reis, M., Vaughn Rouhana, L., Sadeque, A., Koga, L., Clough, S.J., Calla, B., Duarte de Oliveira Paiva, P., Korban, S.S. 2019. Genome-wide expression of low temperature response genes in Rosa hybrida L.. Plant Physiology and Biochemistry. 146:238-248. https://doi.org/10.1016/j.plaphy.2019.11.021.
Zucchi, M.I., Cordeiro, E., Allen, K.C., Lamana, L.M., Viana, J., Brown, P.J., Omoto, C., Pinheiro, J., Clough, S.J. 2019. Patterns of genome-wide variation, population differentiation and SNP discovery of the red banded stink bug (Piezodorus guildinii). Scientific Reports. 9:14480. https://doi.org/10.1038/s41598-019-50999-z.
Zucchi, M.I., Cordeiro, E., Wu, X., Marise Lamana, L., Brown, P.J., Manjunatha, S., Gomes Viana, J., Omoto, C., Pinheiro, J., Clough, S.J. 2019. Population genomics of the neotropical stink bug, Euschistus heros: The most important emerging insect pest to soybean in Brazil. Frontiers in Genetics. 10:1035. https://doi.org/10.3389/fgene.2019.01035.
