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ARS Home » Pacific West Area » Pullman, Washington » Plant Germplasm Introduction and Testing Research » Research » Research Project #434246

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

Location: Plant Germplasm Introduction and Testing Research

2022 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
The Temperate-adapted Forage Legume (TFL) genetic resources program is an important component of the genebank in Pullman, Washington, and operates at the USDA, ARS worksite in Prosser, Washington. The project focuses on acquiring, maintaining, characterizing, evaluating, and distributing alfalfa, clover, trefoil, and their wild relative germplasm accessions. In addition, the project aims to maintain publicly accessible accession-associated documentation in the USDA-developed and globally used Germplasm Resources Information Network (GRIN)-Global database. In support of Sub-objectives 1A and 1B, which focus mostly on service, many of the plant germplasm collection accessions (entries) were grown out to produce seed for restocking (regeneration). A total of 109 accessions were established for seed regeneration including 58 Medicago, 42 Trifolium, six Lotus, and three Acmispon species accessions. Due to low seed recovery from prior year regenerations efforts, 31 accessions were carried over for a second year of seed increases. We collected and captured many traits for accessions being regenerated during the year by scanning and generating digital images for flowers, pods, and seed. For the 2021 calendar year the total number of germplasm distributions was down, especially to international requesters, due to the impacts of the COVID-19 Pandemic and because the National Plant Germplasm System (NPGS) implemented a decision-making process to limit distributions to non-research requestors. Despite this, a total of 4,838 TFL accessions seed packets were distributed in 108 orders to 75 cooperators. In 2021, 11% of the order requests and subsequent shipments were from/to international cooperators. All threshed, cleaned and insect-free seed from 2021 increases was submitted to Seed Storage and Database Manager to be included in the Pullman, Washington, cold storage facilities and made available for distribution late in the calendar year. All passport and associated information including hundreds of voucher images were provided to technical personnel for loading into the GRIN-Global database. Eleven native clover and North American trefoil/deervetch accessions were added to collections from the Seeds of Success collaboration. In support of Sub-objective 1C, project personnel continue to monitor potential unwanted gene flow, or movement of pollen by insects, from genetically engineered (GE) herbicide resistant commercial alfalfa to field regeneration site. In 2022, eight (four newly established in 2022 and four from previous years) sentinel plots were included to monitor potential gene flow and possible contamination. These plots are located on each of the regeneration field corners and are being established with GE-tested/GE-free ‘Vernal’ alfalfa cultivar plants. Seed harvested from these plots is destructively sampled/tested for the unwanted transgene at the end of the season. Four new sentinel plots were established at the research unit's Central Ferry farm to monitor gene flow of transgenic traits there as a potential alternative site for regenerations. In addition, we are testing conventional commercial alfalfa seed lots being used to establish new hay fields on the Prosser experimental farms to establish baseline adventitious presence levels and avoid any high-level transgene contaminated seed lots. More than 36,000 seeds were tested from the ten sentinel plots around regeneration fields in 2021. Positive results were discovered in seed from two out of the ten uncovered sentinel plots, indicating a low level of gene flow. Continued vigilance for timing of installation and proper closure of insect-proof cages continues to be a critical need to avoid possible contamination of alfalfa germplasm regenerations with GE traits. In further progress of Sub-objectives 1B, 2A, and 2B, project personnel worked on establishing several active national and international collaborations. In a collaboration with the researchers in Davis, California, and funded by a USDA National Institute for Food and Agriculture (NIFA) grant, a multi-site replicated alfalfa evaluation field trial was established spring 2021 in Prosser, Washington. A total of 400 individual entries were field transplanted in five-plant plots across two blocks for a total of 4,000 plants. Germplasm being evaluated included wild-collected and landraces plant introduction accessions as well as reference check cultivars. Visually assessed traits like growth habit and leaf/stem ratio as well as yield and quality traits were collected during the fall harvest and will continue throughout 2022. Germplasm being evaluated is to be genotyped with collaborators at Breeding Insight and a genome-wide association study (GWAS) will be conducted to associate markers to important traits. In addition, improved plant selections will be made at the end of the third year and will be combined with collaborators improved germplasm selections of the same original germplasm being evaluated at their sites into improved germplasm for release. All summarized data for traits will be incorporated into the GRIN-Global database and associated with accessions. We have continued to establish working collaborations with public forage breeders (e.g., USDA, academia, non-profit) to evaluate and increase seed of advanced breeding lines. In support of Sub-objectives 1B, 2A, and 2B, a U.S. Alfalfa Farmer Initiative (USAFRI) funded project entitled, “Spring black stem and Stemphylium leaf spot resistance screening in the USDA ARS National Plant Germplasm System’s Medicago sp. genetic resources” wrapped up this fiscal year (FY). From this project a significant number of disease resistant plant selections were made from Standard Check cultivars and from alfalfa plant germplasm screened. Five disease resistant populations corresponding to five fall dormancy groups are being systematically crossed to generate improved populations. Resistant seed from those crosses will be harvested and a second round of selection will be conducted to identify most resistant families. Pooled seed samples from the most resistant crosses will become available as germplasm releases and can be used to breed into commercial cultivars to manage this important foliar plant pathogen of alfalfa.


Review Publications
Nemchinov, L.G., Irish, B.M., Grinstead, S.C., Shao, J.Y., Vieira, P. 2022. Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest. Scientific Reports. 12:8726.
Nemchinov, L.G., Grinstead, S.C., Irish, B.M., Shao, J.Y. 2020. Identification of complete genome sequencing of alfalfa virus S diagnosed in alfalfa plants (Medicago sativa L.) from Washington State, USA. Plant Disease.
Goenaga, R.J., Irish, B.M., Marrero Soto, A.R. 2021. Yield and fruit quality traits of two banana cultivars grown at two locations in Puerto Rico under black leaf streak disease pressure. HortTechnology. 31(6):838-845.