Location: Crop Production and Pest Control Research
2023 Annual Report
Objectives
Objective 1: Identify genetic loci involved in seed oil, protein, and carbohydrate content using forward and reverse genetic approaches, and create genetic combinations that serve as new variability for selection in breeding programs.
Subobjective 1A: Identification of genes implicated in control of seed protein/oil levels.
Subobjective 1B: Alteration of carbohydrate partitioning in soybean seeds.
Subobjective 1C: Combining genes identified by this project to form a basis for improved soybean germplasm.
Objective 2: Evaluate newly identified sources of resistance against Phytophthora sojae, identify candidate Avr gene(s) in the pathogen that are recognized by the new resistance, and design strategies to maintain resistance.
Objective 3: Characterize population structure of Fusarium virguliforme, determine the role of root endophyte community in SDS (soybean Sudden Death Syndrome) resistance, identify key endophytes that can be used for development of new control strategies, and create a SDS genome-wide molecular marker database as a resource for research.
Objective 4: Construct, coordinate, and publish the results of a relevant regional and national variety testing program for soybean that provides timely phenotypic information useful for selection.
Approach
Objective 1: Genes regulating carbohydrate, protein, and fatty acid levels of soybean seeds will be identified using a combination of forward and reverse genetic approaches. Mutants will be evaluated by NIR, GC, and HPLC analysis for multiple aspects of seed composition. Conventional and sequence-enabled mapping techniques will be used to determine gene positions. qRT-PCR will be used to measure the expression of candidate genes during seed development. The best candidate genes will be validated through transformation assays to determine gene impact on seed phenotype.
Objective 2: The performance of resistance genes, including several recently identified, will be evaluated against a large collection of Phytophthora sojae isolates. The best gene pyramiding combination will be recommended for soybean breeding efforts. Genomes and transcriptomes of a subset of isolates will be sequenced to determine how P. sojae evades gene-mediated host resistance, and to identify candidates for uncharacterized Avr genes.
Objective 3: A genome-wide informative microsatellite marker database will be constructed for Fusarium virguliforme using a comparative genomics approach. Identified microsatellite markers will be used to investigate global population structure of F. virguliforme. Prokaryotic and eukaryotic root endobiome of soybean lines susceptible or highly resistant to sudden death syndrome will be characterized using culture and high-throughput sequencing approaches. Endophytic groups associated with SDS resistance will be identified.
Objective 4: Public soybean breeders submit their soybean breeding lines for evaluation of agronomic performance, disease resistance and quality traits. Entries are separated by maturity group and assigned to either the ‘Preliminary Tests’ or the ‘Uniform Tests’. Seeds of each entry, along with those of the standard reference varieties, are packaged and distributed to collaborators throughout the U.S. and Canada for evaluation. In addition, entries will be evaluated at multiple locations in Indiana. Harvested seeds will also be tested for quality traits. Collaborators submit performance data from their locations to ARS after harvest. This data is compiled and analyzed by this research group following established protocols. The results will be published in an annual report book and online.
Progress Report
This is the final report for this project which terminated in May 2023. See the report for the replacement project, 5020-21220-009-000D, “Designing Soybeans with Enhanced Seed Quality, Plant Health Traits and Climate Resilience” for additional information.
Objective 1. Our overall goal was to create and characterize new genetic diversity and identify genes that control soybean seed total protein and oil levels. Over the five-year life of the project, we followed total seed protein and oil content in 106 mutant lines for three or more growing seasons to show that high seed protein or oil levels were reproducible. We performed 72 backcrosses and conclusively determined that 13 lines segregated as dominant high protein, and 19 segregated as recessive high protein, which suggests that there are different genetic mechanisms leading to the composition traits. We observed segregation of the high protein (or high oil) trait in 48 outcrossed populations, and used six of these populations to map the location of the putative new genetic loci. We will continue to evaluate the mutant lines to identify the genes that influence seed total protein and oil in these variants. Currently we are fine mapping one high protein gene located on chromosome 10 that results in a 5-10% increase in total protein, which potentially represents a new gene for high protein. In FY23 we planted seed increases for 115 high protein mutants and altered fatty acid and/or carbohydrate content mutant lines) to enable yield testing and biochemical as well as small- and medium-scale processing studies of these mutants with altered composition. We demonstrated that we can combine up to six alleles to create a soybean with added-value meal trait (low raffinose family oligosaccharides (RFOs)) and the high oleic oil trait. We performed genetic combinations and evaluated seed composition to show that these soybeans include over 80% oleic acid in combination with <0.5% RFOs. The low RFO trait is desirable in soy protein meal, as RFOs are antinutrintional components of the seed and slow growth of non-ruminant animals. These new commodity soybeans add value to both soybean oil and meal for end users. We continue to advance these new genes into higher-yielding germplasm, to determine if there are yield costs associated with the altered composition.
Objective 2. Phytophthora root rot caused by the pathogen Phytophthora sojae has been a major threat to soybean production in the Northcentral region. Host resistance provides our best management tool, but deployed resistance genes (Rps gene) were usually defeated in 7-15 years due to pathogen mutations. ARS scientists in West Lafayette, Indiana, examined pathogen virulence diversity and investigated the molecular mechanism the pathogen uses to evade host surveillance. Over 600 isolates were collected across Indiana. Their virulence was examined on a set of 14 soybean lines, each carrying a different Rps gene. In addition to the usual genes (rps, Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7, Rps8), 3 new genes (RpsUN1, Rps11 and Rps14). These genes were identified by ARS scientists in West Lafayette, Indiana, in collaboration with university faculty and scientists in the soybean breeding industry. Characterization of Rps11 and Rps14 were published . Rps11 was cloned and found to be a 27.7-kb nucleotide-binding site leucine-rich repeat gene. Our results showed that the pathogen virulence was extremely diverse and no Rps gene generated resistance to all isolates. However, Rps8, RpsUN1, Rps11 and Rps14 offered excellent broad-spectrum resistance to a vast majority of isolates. To understand the arms race between the pathogen and its host and the molecular mechanisms the pathogen uses to evade host resistance, 70 isolates were selected for genome and transcriptome (in infected soybean tissue) sequencing. These Phytophthora sojae isolates were selected so that there were multiple isolates that were virulent or avirulent on soybeans expressing each Rps gene. We are mining Phytophthora sojae genomes for avirulence (avr) genes using the hidden markov models and other bioinformatic tools. The goal is to identify avr genes recognized by soybean Rps genes and understand how the pathogen mutates these genes to evade host surveillance.
In addition to P. sojae, a less-known pathogen, P. sansomeana, also causes Phytophthora root rot of soybean. ARS scientists in West Lafayette, Indiana, sequenced the genome of the type strain of this pathogen, 1819B. The mitochondrial genome was published. The nuclear genome is being analyzed in preparation for publication.
Objective 3. ARS scientists in West Lafayette, Indiana, developed a comparative genomics approach to identify genome-wide informative microsatellite markers. Microsatellites are versatile and valuable molecular markers used in many areas of research, such as population genetics, molecular-assisted breeding, genealogy and genome mapping. We first applied this approach to P. sojae (published), and then to Fusarium virguliforme. F. virguliforme caused sudden death syndrome (SDS) in soybean and can be a serious threat when there is a high amount of rain in the growing season. Experimental validation of 29 markers in F. virguliforme showed that this comparative genomics approach was highly accurate, it could eliminate or greatly reduce the need of experimental validation, and thus resolve a labor- and resource-intensive bottleneck in microsatellite marker development. These markers were used to investigate population structure of a collection of 82 F. virguliforme isolates from North and South America where SDS was mostly found. Our data did not provide strong support for the prevailing hypothesis that this pathogen originated from South America but available isolates from there were collected in a narrow temporal and spatial range.
Objective 4. The goal of the USDA Uniform Soybean Test - a community-wide soybean variety testing program designed to help public soybean breeders to overcome the bottleneck of testing advanced breeding lines in multiple environments. As the coordinator, we organized the annual coordination meetings every year to discuss and finalize experimental designs, collected seeds from all collaborators (~20 research groups from the Northcentral United States and Canada), packaged and distributed seeds to all test locations. We also grew field trials in three locations in Indiana. Approximately 500-600 lines were evaluated each year. We collected data on yield and other agronomic traits (flowering time and color, lodging, shattering etc.), their resistance to two major soybean diseases (Phytophthora root rot and soybean cyst nematode), and seed composition (protein, oil and sugar contents and amino acid composition). We also received data from collaborators, compiled and analyzed the data and published annual report books. Hard copies were sent to all collaborators. Electronic copies were published online and freely available. The results from this program are the primary data sources based on which to determine the merits of these advanced breeding lines. From 2018 to 2022, 109 soybean varieties evaluated in the program were publicly released or licensed to private companies.
Accomplishments
1. Mutagenesis produces soybeans with increased protein levels. Soybean protein is an important ingredient in animal feed and human food. Limited genetic variation in soybean is a challenge for crop improvement, as breeders do not have access to a variety of high protein alleles and we lack a mechanistic understanding of what genes can be targeted to improve this trait. ARS scientists in West Lafayette, Indiana, increased soybean diversity using a mutation breeding approach to screen over 5,000 mutagenized lines for total seed protein content. They, identified and characterized 70 soybean lines with the resulting genes that reproducibly confer as much as 10% more seed protein. The researchers went on to identify and sequence the genes conferring increased protein levels. These findings advance our understanding of soybean protein biosynthesis and provide new genes and will provide new strategies and sources of genes for soybean breeders to improve seed protein traits and levels.
2. Development of soybean germplasm DS1260-2 and DS49-142. ARS scientists in West Lafayette, Indiana, together with ARS scientists in Stoneville, Mississippi, developed two soybean germplasm, DS1260-2 and DS49-142, with improved tolerance to mature seed damage. These lines have reduced visual mold, discoloration, wrinkling, and purple seed stain, and improved germination. They will be valuable to soybean growers by improving mature seed quality, reduced dockage, elevated protein content, and seed bean production in hot humid environments.
Review Publications
Hudson, K.A. 2022. Soybean protein and oil variants identified through a forward genetic screen for seed composition. Plants. 11(21). Article 2966. https://doi.org/10.3390/plants11212966.
Detranaltes, C.E., Ma, J., Cai, G. 2022. Phytophthora sansomeana, an emerging threat to soybean production. Agronomy. 12(8). Article 1769. https://doi.org/10.3390/agronomy12081769.
