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Research Project: Management of Temperate-adapted Forage Legume Genetic Resources and Associated Information

Location: Plant Germplasm Introduction and Testing Research

2023 Annual Report

Objective 1: Efficiently and effectively acquire temperate-adapted forage legume genetic resources; maintain their safety, genetic integrity, health and viability; and distribute them and associated information worldwide. Subobjective 1A: Introduce germplasm that fills gaps, is vulnerable or has agronomic potential through plant donations, exchanges and/or explorations. Subobjective 1B: Regenerate temperate-adapted forage legume accessions focusing on low quantity and low viability inventories. Subobjective 1C: Screen for gene flow in germplasm regenerations by assessing adventitious presence of glyphosate resistant seed. Objective 2: Develop more effective genetic resource maintenance, characterization and evaluation methods and apply them to priority genetic resources of temperate-adapted forage legumes. Record and disseminate evaluation and characterization data and digital images via GRIN-Global and other data sources. Subobjective 2A: Using standard and/or new methods, generate and provide access to characterization and evaluation data, collected during in-house regenerations and by leveraging extensive project stakeholder and collaborator networks. Subobjective 2B: Using innovative and diverse molecular marker techniques, estimate genetic diversity and redundancy, identify gaps in coverage and maintain genetic integrity in forage legume germplasm. Objective 3: With other NPGS genebanks and Crop Germplasm Committees, develop, update, document, and implement best management practices (particularly for alfalfa with genetically engineered traits) and Crop Vulnerability Statements for temperate-adapted forage legume genetic resource and information management.

Acquisition of new germplasm will be achieved through collecting and germplasm exchanges. Identifying traits important to the stakeholder community and by comparing representation to current holdings, acquisition targets can be focused. Detailed passport information associated with acquisitions, as well as the germplasm, will become available through the GRIN-Global database. Regenerations will use best management practices to maintain genetic integrity of individual accessions. Prioritization of germplasm to be regenerated will be determined using weighted factors including low seed amounts, viability, age of seed, existence of backup samples, difficulty in regeneration and frequency of requests. Commercial genetically engineered alfalfa is in production and additional measures will be implemented to prevent gene flow. Insect-proof field cages will be placed over all individual accessions from bloom through harvest. Sentinel plots will be used as an effective way of monitoring field site and detecting adventitious presence and possible gene flow. Morphological and molecular techniques will be used to characterize genetic diversity and redundancy, identification of gaps and genetic integrity in the collections. Field and greenhouse-based characterizations and evaluations will be conducted and will focus on disease resistance and agronomic traits using standard test protocols. In addition, digitally captured diagnostic images of floral, fruit, and seed characteristics of regenerated germplasm will be collected. All characterization and evaluation data will be uploaded into the Germplasm Resources Information Network (GRIN-Global) database. As new management techniques are adapted and adopted to increase efficiency and are implemented to secure genetic integrity of germplasm, the standard operating procedures manual will need to be periodically updated. Updated Crop Vulnerability Statements (CVS) will also be developed in consultation with stakeholder community for the major crops managed by the project.

Progress Report
This is the final report for project 2090-21000-026-000D. This project is being combined with the expiring project 2090-21000-032-000D. For additional information, see the new project report 2090-21000-037-000D. This progress report provides a summary of the progress made for all of the objectives and sub-objectives over the life of the project. In support of Sub-objectives 1A and 1B, which mostly address service efforts, the temperate-adapted forage legume (TFL) program distributed just over 26,000 seed lots corresponding to 7,814 accessions during the five-year period between calendar years 2018-2022. ARS researchers in Pullman, Washington, distributed 565 orders to researchers and educators nationally and internationally. Although requests and distributions slowed during the COVID 19 Pandemic, demand for plant genetic resources from the TFL program prior to and since the pandemic is high. Over the same five-year period the program has regenerated an average of approximately 150 unique germplasm accessions per year, focusing on accession seed lots with low stocks or low viabilities. All regeneration activities involve the use of managed insect pollinators in isolation cages, followed by harvest, threshing and meticulous cleaning of seed lots for storage and distribution. During accession regenerations, program staff collected characterization data and generated digital scanned images to upload into the publicly accessible Germplasm Resources Information Network (GRIN)-Global database. This data will aid stakeholders in their online research and selection of germplasm. Collections were expanded with donations and associated information from plant breeding programs or by integrating U.S. native crop wild relatives incorporated through the Seeds of Success program. Additions represented unique genetic backgrounds that strategically filled gaps in the collection. In support of Sub-objective 1C, the program has instituted a gene-flow monitoring program focused on assessing the threat of introducing deregulated transgenic traits (e.g., glyphosate herbicide resistance) from commercial alfalfa grown in the area. The establishment of alfalfa in uncovered sentinel plots on the field periphery allows native insects to pollinate the plants. Harvested seed from these plots is tested for incursion of a transgenic trait or adventitious presence (AP) of transgenes. The covered cage regeneration method was also tested to ensure the insect-proof cages were preventing gene flow. This was done by establishing alfalfa in covered plots with managed insect pollinators, following standard practices, and testing for AP for several years. As no positive AP was ever detected in covered plots, we are satisfied our method prevents transgene contamination of TFL plant genetic resources. In contrast, low levels of AP have been detected in seed lots tested from uncovered sentinel plots every year, indicating transgenic gene-flow to field sites. Project personnel prioritize covering plots of alfalfa and other related species early to prevent gene-flow, AP, and seed lot contamination. In support of Sub-objective 2A, the TFL program completed a U.S. Alfalfa Farmer Initiative (USAFRI) funded project focusing on alfalfa spring black stem and leaf spot (SBS) resistance screening of Medicago spp. genetic resources. The disease is caused by a foliar fungal pathogen and commercial cultivars currently lack resistance. The project optimized inoculation and evaluation protocols, and systematically screened approximately 4,000 lines including alfalfa standard check cultivars, domesticated germplasm, and wild relative accessions. New methods were optimized and are proposed for evaluation with this pathogen in the published standard test protocol. All summarized trait data will be added to GRIN-Global database and associated with accessions. ARS researchers determined the host range within the Medicago genus and identified alfalfa crop wild relatives with increased disease resistance. Disease resistant plants were selected for crosses to develop improved germplasm, including crosses to additional disease resistance selection rounds. Agronomic performance testing is being planned for eventual release of improved germplasm. In support of Sub-objective 2A, a subset of 105 annual medic (Medicago spp.) accessions belonging to 10 species were selected for a diversity assessment study. These accessions were originally assembled from the Crimean region of the Ukraine to fill gaps in coverage in the collections. Many of these annual medics are important forage crops (e.g., M. polymorpha - burr clover) and are wild relatives of cultivated alfalfa that can be used for crop improvement. The accessions were phenotypically and genotypically characterized to determine genetic diversity and population structure and taxonomic identity. Germplasm was grown in the field for two consecutive years in single plots with 10 representative plants per accession. A set of 25 international standard descriptors for annual medics was used to assess highly heritable traits. Phenotypic descriptors showed consistency among replicate plants within accessions, some variation across accessions within species, and evident distinctions between species. The descriptor traits aided in taxonomic identity of a few accessions that were likely mislabeled. Phenotypic data has also shown distinctiveness of Crimean accessions and, along with genotyping, will help determine what germplasm to include in permanent collections. Final summarized trait data will be associated with accessions in GRIN-Global. In support of Sub-objectives 2A and 2B, collaborative projects are generating, evaluating, and selecting improved alfalfa germplasm in prebreeding efforts. Supported by a USDA , National Institute of Food and Agriculture (NIFA) grant and in collaboration with Breeding Insight (BI) and the University of California, we evaluated a large subset of 400 accessions, 10 plants each, in a multi-site (Washington and California) replicated field trial. With support from BI, phenotypic, agronomic and forage quality traits were collected electronically using the Field Book application. All accessions are being genotyped with a recently developed platform for alfalfa. Genotypic and trait evaluation data will be used in genome-wide association studies (GWAS). Germplasm that is determined to be locally adapted will be selected and crossed with plant selections from California, and resulting populations will be released to broaden narrow the genetic base of commercial alfalfa cultivars. All summarized data for traits from these evaluations will be incorporated into GRIN-Global and associated with accessions. Due to our project expertise, infrastructure, and ideal growing conditions, the TFL program has been collaborating with public forage breeders (e.g., USDA, academia, non-profit) across the nation to evaluate and increase seed of advanced breeding lines to publicly release improved germplasm. In support of Sub-objectives 2A and 2B, ARS researchers in Pullman, Washington, collaborated with scientists at Loyola University in Chicago, Illinois, to characterize clover (Trifolium spp.) germplasm by sequencing of DNA barcodes to correctly identify species/taxa. Work has focused on identifying which DNA barcodes are optimal for clover species while trying to correctly assign taxonomy when unknown, and/or where mixes, misidentification or mislabeling might have occurred. Voucher DNA barcode sequences were developed for 98 accessions representing 20 species and deposited in GenBank to serve as references for future studies. In the process, correct identify was proposed for 10 accessions previously only identified to the genus level and for 14 accessions where misidentification or mislabeling had occurred. All germplasm accessions identified to species for the first time and/or identified using the DNA barcodes were grown in greenhouses for botanical validation. These efforts improve collection quality and provide stakeholders with increased access to correctly identified plant germplasm. In support of Objective 3, a project-specific manual of operating procedures (MOP) document outlining daily processes followed in implementing the current project is routinely updated. This MOP outlines the best management practices and includes any new techniques or approaches undertaken to execute effectively and efficiently the TFL germplasm project. The MOP updates include the use of clonal propagation techniques when low numbers of plants (<100) are available for regenerations, the option of using in vitro/laboratory germination for low-seed number and/or viability inventories when establishing plants in greenhouses for eventual field regenerations, and the use of several species of bumblebees for optimized pollinations in clover species. Another addition to the MOP is the use of sentinel plots for monitoring gene flow and AP of genetically engineered traits to the alfalfa field regeneration site and references to both the “ARS Procedures & BMPs for GE Traits in Plant Germplasm and Breeding Lines” as well as to the USDA-ARS Policies and Procedures (603.0) entitled “Management of Genetically Engineered (GE) Traits in Plant Germplasm and Breeding Stocks”. Both official documents provide detailed information and guidance on how the NPGS will meet its mission while continuing to ensure genetic integrity of the collections in the presence of continued deregulation and production of agricultural crops with GE traits.