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

2023 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
This progress report provides a summary of the first year of this newly established project 2090-21000-037-000D which was replaced by expired projects, 2090-21000-026-000D and 2090-21000-032-000D, which were combined into this new project. Supporting Sub-objective 1A, the Temperate Forage Legume (TFL) program received a donation of 195 barrel medic accessions from French collaborators. This collection of a model plant species represents a subset of genetically diverse accessions that has been genetically characterized. The Phaseolus (Bean) program has increased the diversity and collection holdings of all 3 subspecies of wild kidney bean, important crop wild relatives (CWR). Collection trips to eight locations in the states of Georgia and Florida by ARS program personnel and collaborators allowed collections that filled a gap in collection coverage for the subspecies, as well as a geographic gap from the southeast United States. Every year, expired intellectually protected germplasm is released to corresponding programs, with 87 accessions incorporated in CY 2023. In the past year, 585 accessions were added to the Pullman-based collections originating from the Bureau of Land Management (BLM)-led Seeds of Success (SOS) program. All germplasm acquired (donations, transfers, and explorations) increase needed genetic diversity by filling gaps in coverage and are made readily available. Supporting Sub-objective 1B, the TFL program continues to focus on regenerating germplasm accessions only identified to the genus level and accurately identify them taxonomically, using highly heritable phenotypic traits. From this year’s plantings, approximately 10 accessions were correctly identified to the species level and records modified in the Germplasm Resource Information Network (GRIN)-Global to reflect this. In a similar approach the Phaseolus (Bean) program continues regenerations of older accessions while correcting misidentified species based on phenotypes and has correctly identified 10 misidentified lima bean accessions. Complete and accurate identification of accessions held in the collections incentivizes their use by stakeholders. Across all curatorial programs in FY 2023, more than 30,000 items (e.g., seed packets) of more than 20,000 accessions were distributed to stakeholders nationally and internationally, with significant distributions (>1,000) to plant breeding organizations (both public and private). Supporting 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 year, 2,211 accessions were scheduled to be regenerated on three research farms in Pullman, Central Ferry and Prosser, Washington. All germplasm is queued for regeneration based on low seed stock and/or seed viabilities below critical thresholds, or because clonal stock needs replanting. Supporting Sub-objective 2A, the TFL program continues to evaluate approaches to germinate, establish and increase seed for cultivated clover crop wild relatives (CWR). In collaboration with researchers at Lewis and Clark State College, several stratification regimes were evaluated on big-headed cover. Optimal germination was observed for seed treated with cold moist stratification for four weeks. Germinated seedlings were used to establish field regeneration plots in Prosser, Washington. Unfortunately, no transplants survived, demonstrating how important it is to optimize field plant establishment. The Phaseolus program worked to increase regeneration efficiency and seed quality by testing direct threshing of greenhouse produced plots instead of sequential hand-picking mature pods, decreasing time between harvest and cold room storage, 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. For Sub-objective 2B, the TFL program finished field evaluation of alfalfa germplasm accessions established in a 4,000-plant trial. Data included highly heritable phenotypic traits, agronomic and quality traits and was collected over multiple harvests and years. The data will be shared with project collaborators at Breeding Insight (BI) in Ithaca, New York, and University of California, Davis to conduct genome-wide association studies (GWAS). The Phaseolus program initiated field characterizations to evaluate growth habit and photosensitivity, which are important traits for breeders and difficult to phenotype in the greenhouses where seeds are regenerated. The program is also collaborating on a Specialty Crops Research Initiative (SCRI) lima bean grant to phenotype the entire available collection (700 accessions), which have been established in the field for data collection in 2023. In addition, and during the yearly regeneration efforts, curatorial program’s personnel continued to collect voucher 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. For Sub-objective 2C, the TFL program is working with collaborators at BI to develop a single nucleotide polymorphism (SNP) genotyping platform in alfalfa to genotype a set of 400 alfalfa germplasm accessions. SNP data, along with field phenotypic and agronomic data, will be used to conduct GWAS. The TFL program is collaborating with a group of public clover breeders to increase genomic resources by growing and contributing CWR germplasm samples for whole-genome sequencing in a Joint Genomes Institute grant. In the Agronomy program, 864 safflower accessions were genotyped by sequencing (GBS) in collaboration with researchers at West Virginia State University. The initial sequencing reads have been filtered for quality, aligned, and a total of 965,850 SNPs uniformly spread over the 12 chromosomes were identified in the dataset. In the Cool Season Food Legumue (CSFL) program, SNP data were collected for 480 lentil accessions. Some were collected with GBS and some with higher density sequencing; a final dataset will be created with imputation to have the most high-quality SNPs on all 480 lines. In the SCRI lima bean grant collaboration described earlier, the Phaseolus program has collected leaf material for the 700 accessions for genotyping. 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 including a broad geographic diversity. Several potential gene candidate loci have been identified that will aid in population genetic analyses of the larger lettuce collection. Genotypic data generated for collections help understand population structure and relationships in accessions, and often can be used to link traits of interest for breeding. For Sub-objective 3A, the TFL program continues to develop improved alfalfa and clover germplasm. The materials being developed from the NPGS collections are being selected for several traits, across locations, and cropping systems. Spring blackstem resistant alfalfa maternal-half sib populations in five distinct fall dormancy groups were developed from resistant selections and subsequent crosses. These populations will be screened for disease reaction and the most resistant families will be bulked for further agronomic evaluation and release. Following three years of field evaluation, 120 plant selections were made from a 4,000-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 UC Davis. These materials will be further evaluated in recurrent selection programs for eventual release. As part of the lima bean SCRI project the Phaseolus 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, seventy-five 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. Supporting Sub-objective 3B, the CSFL program conducted a GWAS for pea seed protein concentration collected from field grown peas over three years and identified over 20 SNP markers significantly associated with protein levels. The 10 SNPs that were identified in more than one environment and with a large phenotypic effect following the GWAS analysis were prioritized for marker development and converted to a more straightforward kompetitive allele specific PCR (KASP) assay and will be validated in field grown materials in the next year. Development of markers linked to important agricultural traits is useful in increasing efficiencies in plant breeding. For 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 of PAST. The PAST tool optimized for different mating strategies in plants will improve precision in marker trait association studies.

Accomplishments
1. Drought tolerant lentil genetic resources identified as sources for breeding. Lentils are an important pulse crop that provides great sources of protein and other important nutritional components in healthy diets. Climate change continues to challenge agricultural production with traits for abiotic stressors often coming from plant genetic resources collections. ARS researchers in Pullman, Washington, along with collaborators screened through a significant number of samples in the lentil collection using high-throughput reflectance methods and multispectral radiometers and identified several accessions with drought tolerant attributes. These accessions can be used in breeding resilient drought-tolerant crops and contribute to sustainable agricultural production, which will benefit both growers and consumers.

2. Multi-trait genomic selection approach could accelerate breeding timelines. Improved techniques and tools are needed to gain efficiencies in the plant breeding process. Researchers at North Dakota State University and ARS researchers in Fargo, North Dakota, and Pullman, Washington, used a multi-trait genomic selection approach using artificial intelligence and machine learning processes to improve predictive nutritional breeding values in pea germplasm from genotypic data and sparse, but correlated, phenotypic datasets. Results showed improved predictive ability of this approach increasing the effectiveness in breeding methods. The technique also indicated that a reduction in costs could be realized by minimizing the time-consuming phenotypic data collection process used traditionally by plant breeders. Nutritionally improved pulses like pea, deliver plant-based proteins, minerals and vitamins with potential to improve consumer diets.

3. Optimizing genotyping platform for annual medic plant genetic resource collections. Annual medics are close relatives of alfalfa and important as forage legumes, cover crops, and green manures. ARS researchers at the USDA ARS National Plant Germplasm System, in Pullman, Washington, conserve and promote the use of significant annual medic plant genetic resource collections. To estimate coverage and completeness in the annual medic collection, a partnership was established with Breeding Insight in Ithaca, New York, to use a novel genotyping platform recently developed for assessing diversity in alfalfa. The platform worked well and aided in resolving relationships among samples in the collections and in precise taxonomic identification. These efforts acted as a proof-of-concept for the platform, demonstrating its practicality in characterizing annual medics in these evaluations and showing that the technology could be extended to other portions of the existing NPGS collections and/or to the larger scientific community.

U.S. DEPARTMENT OF AGRICULTURE

Plant Germplasm Introduction and Testing Research: Pullman, WA