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.
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 as well as database-maintaining all associated documentation. All of the objectives and sub-objectives of this 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, a total of 160 accessions were planted for seed increase (regeneration) including 51 Medicago species, 86 Trifolium species and 19 Lotus species. Due to low seed recovery from the prior year regenerations efforts, 35 accessions were carried over for a second-year increase. Traits for many of the accessions regenerated during the year were collected and captured by scanning images for flowers, pods and seed. For the 2019 calendar year a total of 4,509 alfalfa, clover, trefoil and their crop wild relative items were distributed in 146 orders to 119 cooperators. Just over 13% of the order requests, and subsequent shipments, were from or to international cooperators. All threshed, cleaned and insect-free seed from 2018 increases was submitted to Seed Storage Manager to be included in the Pullman cold storage and made available for distribution late in the calendar year. In addition, in 2019 the program received a number of improved, cold-hardy alfalfa and alfalfa hybrids. These materials will be useful as parents for developing new alfalfa varieties with improved cold-hardiness that can better survive harsh winters. In support of Sub-objective 1C, the threat of gene-flow from genetically engineered (GE) alfalfa to the temperate forage legume collection continues to be high and is only expected to become more severe as a second genetically engineered trait in alfalfa, low-lignin content, becomes commercialized. To monitor this potential gene-flow a total of 14 sentinel plots were included in the regeneration field in 2019. These plots are established with GE-tested/GE-free alfalfa plants (variety “Vernal”). Seeds harvested from these plots were tested for adventitious presence of the unwanted glyphosate resistance transgene. From the seven covered ‘Vernal’ alfalfa variety sentinel plots around regeneration fields in 2019, more than 25,000 seeds were tested and no adventitious presence (AP) - finding the transgene where it was not wanted - was detected. Two out of the seven uncovered plots showed positive results in testing, indicating gene-flow to the regeneration site. These results suggest that our isolating procedures using “seed cages” are effective at eliminating contamination by pollen from genetically engineered alfalfa. Significant progress was made on Sub-objective 2A, which focuses on screening alfalfa germplasm for resistance to fungal disease. Spring black stem caused by Phoma medicaginis, and Stemphylium leaf spot caused by Stemphylium spp., which are important alfalfa foliar fungal plant pathogens for which good disease resistance is not available in commercial cultivars. We completed initial disease screenings and have identified resistant alfalfa materials. These materials can be used as parents to develop new alfalfa varieties with enhanced resistance to fungal diseases. Considerable progress was made on Sub-objective 2B, which is focused on systematic characterization of an annual medic (Medicago spp.) collection originating from the Crimean Peninsula of Ukraine. The collection consists of close to 100 samples representing ten different lineages. These samples and an additional 100 reference samples were evaluated for more than 20 traits associated with plant appearance, growth, and development. Prior to field establishment, DNA was extracted from all samples. DNA fingerprints will be developed for all samples and used in conjunction with field traits to determine how to integrate these Crimean samples into existing plant diversity collections. In support of Objective 3, a Manual of Operating Procedures (MOP) used to document daily processes followed in the current project, is being routinely updated. Recent additions to the manual include 1) the use of sentinel plots for monitoring gene flow and unwelcome presence of genetically engineered traits to the alfalfa field regeneration site; 2) the addition of sentinel plots at the Unit’s Central Ferry research farm for monitoring gene flow, and 3) references to both the “ARS Procedures & Best Management Practices for Genetically Engineered Traits in Plant Germplasm and Breeding Lines” developed for National Program 301, as well as to the USDA-ARS Policies and Procedures (603.0) entitled “Management of Genetically Engineered Traits in Plant Germplasm and Breeding Stocks”. Both official documents provide detailed information and guidance on how the National Plant Germplasm System (NPGS) will ensure genetic integrity of the collections in the presence of continued deregulation and production of agricultural crops with GE traits.
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