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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

2021 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 Plant Germplasm Introduction and Testing Research Unit (PGITRU) 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 a USDA-developed and globally used plant genetic resources (Germplasm Resources Information Network [GRIN]-Global) database. All of the objectives and sub-objectives of this service and research address Problem Statement 2A (Plant and microbial genetic resource and information management) of Component 2 (Plant and microbial genetic resource and information management) of the National Program 301, Plant Genetic Resources, Genomics, and Genetic Improvement (2018-2022). 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. As well, a total of 110 accessions for seed increase (regeneration) including 47 Medicago, 51 Trifolium, four Lotus, and four Acmispon species accessions were established. In addition, four new alfalfa sentinel field plots were established and harvested for detection of potential unwanted transgenic traits (i.e., glyphosate herbicide resistance) during the 2021 growing season. Due to low seed recovery from prior year regeneration efforts, 34 accessions were carried over for a second-year increase. We collected and captured many traits for accessions being regenerated during the year by scanning and generating voucher images for flowers, pods, and seed. For the 2020 calendar year the total number of germplasm distributions was down, especially to international requesters, due to the impacts of the COVID-19 Pandemic. Despite this, a total of 4,800 forage legume accession seed packets in 90 orders to 69 cooperators were distributed. In 2020, 9% of the order requests and subsequent shipments were from or to international cooperators. All threshed, cleaned, and insect-free seed from 2020 increases was submitted to the Seed Storage 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 Germplasm Resource Information Network (GRIN) Global database. In addition, a single accession of barrel medic (Medicago truncatula), used as a genetic tool in functional genomics research, was donated and added to the collection. 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 our field regeneration site. In 2020, we included ten sentinel plots to monitor potential gene-flow and possible adventitious presence. Seed harvested from these plots is destructively sampled/tested for adventitious presence (finding of the unwanted transgene) of herbicide resistance at the end of the season. More than 36,000 seed from the ten sentinel plots around regeneration fields in 2020 were tested. Positive results were found in seed from four out of the seven uncovered sentinel plots, indicating gene-flow to the regeneration site. 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 with GE traits. In support of Sub-objective 2A, a project that focused on developing optimized screening protocols and identifying sources of resistance to alfalfa spring black stem and leaf spot in germplasm collections was completed. Where this disease occurs, yield and quality can be severely reduced and currently no good resistance exists in commercial cultivars. Published protocols were optimized and improved by proposing modifications to lower spore concentrations and substituting more resistant cultivars as references. In addition, the host range was more precisely defined within the genus by screening through diverse alfalfa related Medicago plant materials. Lastly, a large subset of alfalfa plants was selected from more than 2,800 germplasm accessions screened for their low disease reaction rating (<1.5). These plants are being used for development of improved disease resistant populations. The modifications to the standard protocols are being published and will be available to broader scientific community to be used when working with this disease. Advanced alfalfa germplasm releases can be used to improve commercial cultivars and manage this important foliar disease of alfalfa. In support of Sub-objectives 1B and 2A, project personnel also established several national and international collaborations. Collaborations were advanced with forage breeders at the University of California, Davis, and ARS location in Madison, Wisconsin, on selecting USDA-National Plant Germplasm System (NPGS) alfalfa germplasm that is regionally adapted. In this collaboration, seed from alfalfa selections and crosses from five collaborators in northern alfalfa production regions (California, Wisconsin, and New York and Alberta and Quebec, Canada) was pooled and increased in Prosser, Washington. These increased seed selections will be tested in large field trials for improved germplasm release in the next few years. In addition, a large 400 accession alfalfa space plant nursery has been established and first year data will be collected in the fall of 2021. In this evaluation, germplasm that is locally adapted is being selected for improved performance at the Prosser, Washington, site. This improved germplasm will be selected and eventually used by commercial companies to increase the narrow genetic base of commercial alfalfa cultivars. Project personnel also began working with ARS scientists in Madison, Wisconsin and forage breeders in Cornell University in Geneva, New York, on testing new and improved ways to increase seed of advanced clover populations. Testing has included the establishment and regeneration of crimson clover (Trifolium incarnatum), a species not previously grown at the Prosser site. In addition, evaluation began on different pollinator bee species including honeybee as well as native and non-native bumblebee species for pollination efficiency of red clover (Trifolium pratense).

1. Sources of improved disease resistance identified in alfalfa. Spring blackstem and leaf spot is an important disease, causing yield and quality losses in alfalfa. Currently, effective disease resistance is lacking in commercial cultivars and chemical control is not economically feasible. Therefore, a need exists to identify potential sources of resistance in germplasm than can be incorporated in modern commercial cultivars. In a collaboration between ARS researchers in Prosser, Washington, and Saint Paul, Minnesota, improved methods were developed to screen alfalfa for important diseases and sources of resistance were identified to spring blackstem and leafspot disease. These resistant plants are being used to develop new populations from which improved commercial alfalfa varieties can be derived.

Review Publications
Irish, B.M., Greene, S.L. 2021. Germplasm collection, genetic resources, and gene pools in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. p.43-64.