Project : USDA ARS

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Research Project: Genetic Resource and Information Management for Pulse, Temperate Forage Legume, Oilseed, Vegetable, Grasses, Sugar, Ornamental, and Other Crops

Location: Plant Germplasm Introduction and Testing Research

2024 Annual Report

Objectives
Objective 1: Acquire, distribute, and maintain the safety, genetic integrity, health, and viability of priority pulse, temperate forage legume, oilseed, vegetable, turf and forage grass, sugar, ornamental, and medicinal plant genetic resources and associated descriptive information. Sub-objective 1.A: Acquire samples and associated passport information of priority plant genetic resources (including crop wild relatives) from the U.S. and/or other countries to fill current gaps in NPGS genetic resource collections. Sub-objective 1.B: Classify, conserve, and distribute PGITRU plant genetic resources and their associated information. Sub-objective 1.C: Regenerate accessions of priority plant genetic resources, emphasizing accessions with low germination, few seeds in storage, or those not yet backed-up at secondary sites. Objective 2: Conduct research to develop genetic resource maintenance, evaluation, or characterization methods and, in alignment with the overall NPGS Plan, then apply them to priority pulse, temperate forage legume, oilseed, vegetable, turf and forage grass, sugar, ornamental, and medicinal plant genetic resources to avoid backlogs in plant genetic resource and information management. Sub-objective 2.A: Conduct research to identify storage and quality regeneration conditions ideal for priority crops and wild relatives. Sub-objective 2.B: Evaluate germplasm accessions for priority agronomic and horticultural traits (e.g., nutritional) and biotic and abiotic stresses. Incorporate evaluation data into the GRIN-Global and/or other databases. Sub-objective 2.C: In collaboration with university and industry partners, apply genotypic characterization techniques (e.g., next generation sequencing) and platforms (e.g., arrays) to selected crop accessions to estimate genetic diversity, relationships, and population structure, and identify gaps in the genetic coverage of the collection. Incorporate characterization data into the GRIN-Global and/or other (e.g., SciNet, NCBI, etc.) databases. Sub-objective 2.D: With other NPGS genebanks and CGCs, develop, update, document, and implement best management practices and Crop Vulnerability Statements (CVS) for legume, oilseed, vegetable, turf and forage grass, sugar, ornamental, and medicinal genetic resource for which they are lacking. Objective 3: Breed genetically-enhanced germplasm that broadens the diversity available for improving selected crops by incorporating superior traits from cultivars, landraces, and wild relatives into adapted genetic backgrounds and gene pools. Sub-objective 3.A: Conduct collaborative crossing and selection programs to breed agronomically improved and disease-resistant germplasm. Sub-objective 3.B: Through genomic and field evaluation data analyses, identify genetic markers associated with quality traits and resilience to biotic and abiotic stresses for application to crop genetic enhancement. Sub-objective 3.C: Develop genetic mapping tools and resources for elucidating and validating the genetic basis of economically important traits for incorporation into genetic enhancement programs.

Approach
Conserve, regenerate, evaluate and distribute ~100,000 accessions of cool season food and forage legumes, grasses, common beans, oilseeds, vegetables, beets, ornamentals, medicinal crops and related and native wild species, and associated information according to the National Plant Germplasm System Distribution Policy and the established protocols and procedures. Keep our active plant genetic resource collections in the seed storage facilities with adequate conditions for proper conservation of seed samples for short and medium term storage and for people entering the storage space to take samples for distribution and for viability tests. Monitor seed viability by periodic germination tests at variable intervals depending on the species. Ship high quality seed samples to National Laboratory for Germplasm Resources Preservation at Ft. Collins, Colorado and the Svalbard Global Seed Vault in Norway for long-term security back-up. Try to address backlogs in regeneration and data entry into the Germplasm Resources Information Network (GRIN)-Global where these backlogs exist. Conduct collaborative plant collection trips and germplasm exchange to acquire samples to fill gaps in NPGS collections, and to supply critically needed traits to support current and future breeding and research. Evaluate the phenotypic variation of economic traits of specialty crops independently or collaboratively. Use laboratory equipment to characterize major nutritional components of food crop germplasm such as using near infrared (NIR) spectroscopy to quantify the major nutritional component content of food legume genetic resources. Apply existing and newly developed genomic tools and technologies such as the Next Generation DNA sequencing to characterize genetic diversity, phylogenetic relationship and marker-trait association of priority crop collections. Upload characterization/evaluation data into GRIN-Global and/or other databases. Conduct research on best methods to regenerate wild species in the collections, including germination and pollination requirements. Survey production fields, identify pathogens causing emerging diseases with morphological, cultural and molecular techniques, investigate interactions among these host plants and their pathogens, and devise and apply pathogen management strategies to maintain the health of the assigned genetic resources. With collaborators, create new crosses with genebank materials to create segregating material that will be of use to breeders. Create new bi-parental populations or diverse panels for use in identifying new genes controlling crop traits of interest. Validate and make markers linked to genes controlling useful crop traits easy to use by public breeders. Publish research results and release segregating or improved germplasm, and useful genetic markers, to the user community. Update the pertinent section of Operations Manual with reference to changes in collection holdings, management technologies and practices, diagnostic procedures, roles of personnel and any other relevant changes. Work with relevant crop germplasm committees to update the Crop Vulnerability Statements of the crops under our management.

Progress Report
Supporting Sub-objective 1A, the Bean program collected seed of wild bean species from 14 populations in southern New Mexico. Seeds collected are available for distribution and include 10 wild tepary with potential heat and drought tolerance. Each year expired intellectually property protected germplasm is released to corresponding programs, with 100 accessions incorporated in FY 2024. In the past year, 715 accessions were added to the Pullman-based collections originating from the Bureau of Land Management-led native Seeds of Success program. In addition, 122 accessions are held from collections made by both U.S. Fish and Wildlife Service and National Park Service. All germplasm acquired (donations, transfers, and explorations) increase needed genetic diversity by filling gaps in coverage and are made readily available. In support of Sub-objective 1B, each of the curatorial programs continues to correct taxonomic identification of misidentified, mislabeled, mixed or incompletely identified accessions in the collections. This is often done during seed regenerations using highly heritable phenotypic traits, but also more recently via genetic markers and genomic approaches. From this year’s plantings, approximately eight, five and 46 accessions were correctly identified to the species level in the forage legume program, bean, and horticultural crops programs, respectively. All records have been updated in Germplasm Resource Information Network (GRIN)-Global. Across all curatorial programs in FY 2024, more than 36,187 items (e.g., seed packets) of more than 25,467 accessions were distributed to stakeholders nationally and internationally, with significant distributions (9,265) to plant breeding organizations (120 unique requestors, both public and private). Complete and accurate identification of accessions held in the collections incentivizes their use by stakeholders, as evidenced by significant distributions. Under Sub-objective 1C, genebank curatorial programs continue to focus on the core mission of long-term preservation by regenerating both seed and clonally propagated accessions using optimized and crop-specific best management practices. This fiscal year, 2,128 accessions were scheduled to be regenerated on three research farms in Pullman, Central Ferry, and Prosser, Washington. All germplasm in collections, most of which is irreplaceable, is queued for regeneration based on low seed stock and/or seed viabilities below critical thresholds, or because clonal stock needs replanting. For Sub-objective 2A, all curatorial programs continue to evaluate approaches to germinate, establish, and effectively increase germplasm, especially for wild relatives. This year the Forage Legume program compared annual medic seed increases in greenhouse and field and direct seeding vs. transplants to optimize plant establishment of alfalfa wild relatives. The Bean program is working to improve seed production rates from perennial bean species in greenhouses, increase regeneration efficiency and seed quality by testing direct threshing of greenhouse produced plots instead of hand-picking pods, and scarifying and pre-germinating seeds to establish seedlings in accessions with low vigor. Efficiencies gained by researching optimal storage and regeneration techniques allow for the safeguarding long-term the important collections in the genebank. In support of Sub-objective 2B, the Forage Legume program finished evaluation of alfalfa germplasm accessions established in a 4,000-plant field trial. Highly heritable phenotypic traits, agronomic, and quality traits are being used by project collaborators at the University of California, Davis (UCD), to conduct marker-trait associations. 132 plant selections with favorable agronomic traits were clonally propagated and shared with UCD collaborators for crosses and improved population development. The Bean program continues field characterizations of growth habit, development rate, and photosensitivity, traits of importance to breeders and difficult to phenotype in greenhouses where seeds are regenerated. The program is also continuing to collaborate on a Specialty Crops Research Initiative (SCRI) lima bean grant to phenotype the entire available collection (700 accessions) in multiple locations. During annual regeneration efforts, curatorial program’s personnel collected digital images of plant leaves, leaflets, flowers, and fruit/pods to be associated with accessions in GRIN-Global. Detailed characterization and evaluation data focused on traits of interest associated to germplasm collections increases its value and use. Progress on Sub-objective 2C in the Forage Legume program included working with collaborators at Breeding Insight to genotype a set of 400 alfalfa germplasm accessions with a single nucleotide polymorphism (SNP) genotyping DArTag platform. SNP data, field phenotypic, and agronomic data, are being used to conduct genome-wide association studies (GWAS). Whole-genome sequencing will be completed for 56 cultivated clover wild relative accessions in a Joint Genomes Institute grant to increase genomic resources available to public clover breeders. In the Agronomy program, the 965,850 SNPs identified from genotyped by sequencing (GBS) of 864 safflower accessions have been made publicly available on the National Center for Biotechnology Information (NCBI) and the Safflower Genetics Resources websites. In the Cool Season Food Legume (CSFL) program, almost 1.7 million SNPs were identified in a panel of 165 lentil lines, and GWAS is now being used on seed protein and multiple related phenotypes. In the SCRI lima bean grant collaboration described earlier, the Bean program has sequenced 700 accessions. In the Horticultural Crops program, an exome capture approach screening 1,061 genic regions was used to accurately characterize 150 lettuce accessions including material without species designation. A more robust phylogeny for the genus Lactuca was constructed with the data along with identifying several potential gene candidate loci utilizing a random forest model that will aid in population genetic analyses of the larger lettuce collection. For the rhubarb collection, 24 SSR markers were used on 56 clones from the genebank and those collected in Alaska. The data allowed the Horticultural Crops curator to choose unique clones from Alaska to fill gaps in the collection. Genotypic data generated for collections help to understand population structure and relationships in accessions and often can be used to link traits of interest for breeding. For Sub-objective 3A, the Forage Legume program continues to develop improved alfalfa and clover germplasm. Additional spring blackstem resistant alfalfa populations in four distinct fall dormancies are being developed for their disease resistant and promising agronomic traits. These populations will be screened for disease reaction and recurrently selected for disease reaction, agronomic evaluation and release. Following three years of field evaluation, 120 alfalfa plant selections were made from a 4,000-space plant trial. Plants were selected for their persistence and yield and are being used in crosses with selections made of the same evaluated materials by collaborators with UCD. The next generation will be further evaluated in recurrent selection programs for eventual release. As part of a lima bean SCRI project, the Bean program created 11 putative crosses between photoperiod sensitive exotic lines and photoneutral commercial lines. These hybrids will be verified, advanced to F2 and shared with collaborators and the public. In addition, 75 faba bean breeding lines from the CSFL program selected by the now-retired research geneticist were planted in Central Ferry, Washington, for further evaluation. Prebreeding efforts make use of in-house collection expertise and are geared towards incorporating useful traits into improved germplasm for commercialization by interested stakeholders. Under Sub-objective 3B, the CSFL program conducted a GWAS for protein and fat concentration in seeds, and flower color, collected from field grown peas over three years for 487 pea accessions using 114,687 SNPs collected previously. Significantly associated genes and pathways were identified. Researchers identified 15 SNP markers significantly associated with protein levels and 11 associated with fat. Metabolic pathways associated with both traits were also identified involving fatty acids, amino acid and protein metabolism, and the tricarboxylic acid cycle (TCA). These findings will aid breeding of high-protein, diverse pea cultivars, and breeder-friendly molecular marker assays are presented for genes associated with high protein. Thirteen lima bean families created from non-adapted exotic accessions and temperate adapted cultivars were created and are under field evaluation and selection at a university research facility in South Carolina. Development of markers linked to important agricultural traits is useful in increasing efficiencies in plant breeding. In support of Sub-objective 3C, the Pathway Analysis Study Tool (PAST) was optimized to study metabolic pathway analysis of GWAS results in inbreeding species. The PAST tool had been developed for maize, an outcrossing species, and the linkage disequilibrium patterns for inbreeding species is different. A new way to assign SNPs to genes for subsequent assignment to pathways was developed and is in the new beta release version. The PAST tool optimized for different mating strategies in plants will improve precision in marker trait association studies. This was tested in the panel of 487 pea accessions, and successfully identified 27 pathways associated with fat and protein in seeds. As a test case, PAST was used for flower color, and the mechanism causing segregation of purple and white flowers described by Gregor Mendel, was successfully identified.

Accomplishments
1. Upgrades to the Pathway Association Study Tool (PAST) program leads to significant increase in software downloads/use. The software program PAST identifies plant metabolic pathways associated with plant breeding traits of interest. Upgrades in the program have been tested for use with inbreeding species, which were difficult due to complex genetic inheritance. PAST was successfully used with pea, an inbreeding species. This has increased its usefulness in plant breeding, as evidenced by the significant increase recent in software downloads, from an average of 85 downloads per month to an average of 440 per month in the second quarter of 2024. The upgraded software version adds versatility to plant breeders wanting to explore and describe more precisely genic regions conferring specific traits.

2. Taxonomic assignments in lettuce germplasm using modern genomic approaches. In the Horticultural Crops program, an exome capture approach screening 1,061 genic regions was used to accurately characterize lettuce germplasm accessions including material without species designation. The approach used 150 accessions spanning 27 species and included a broad geographic diversity. ARS researchers in Pullman, Washington, constructed a more robust phylogeny for the genus Lactuca was constructed with the data along with identifying several potential gene candidate loci utilizing a random forest model that will aid in population genetic analyses of the larger lettuce collection. Increasing the number of correctly identified accessions and developing genomic approaches to assess diversity in collection makes the lettuce germplasm more useful to plant breeders and other germplasm stakeholders.

U.S. DEPARTMENT OF AGRICULTURE

Plant Germplasm Introduction and Testing Research: Pullman, WA