1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
The Temperate-adapted Forage Legume (TFL) genetic resources program is managed by ARS scientists in Pullman, Washington, and housed at the 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. In support of Sub-objectives 1A and 1B, a total of 156 accessions were planted for regeneration including 48 Medicago species, 76 Trifolium species and 18 Lotus species and 14 sentinel alfalfa plots for adventitious presence transgene detection for the 2019 growing season. Due to low seed recovery from the prior year regeneration efforts, 56 accessions were carried over for a second-year increase. Phenotypic traits for many of the accessions regenerated during the year were collected and captured by scanning and generating voucher images for flowers, pods, and seeds. For the 2018 calendar year a total of 7,688 alfalfa, clover, trefoil, and their crop wild relative items were distributed in 146 orders to 128 cooperators. Just over 14 percent of the order requests, and subsequent shipments, were from/to international cooperators. All threshed, cleaned and chalcid-free seed from 2017 increases was submitted to Seed Storage Manager to be included in the Pullman cold storage and made available for distribution late in the 2018 calendar year. An average of close to 151 grams of seed per accession/plot was achieved for seed cleaned for the 2017 growing season (vs. 300 grams/plot for 2016). All passport and associated information as well as germinations to determine baseline viability (75 accessions tested) and voucher images (391) were provided to technical personnel for loading into the Germplasm Resource Information Network (GRIN) Global database. In addition, a select number of improved, cold-hardy alfalfa and alfalfa hybrids accessions were donated to the program by a retired professor at the University of Wisconsin at Madison. The project also accepted 16 native clover and trefoil taxa from the Seeds of Success project that fills gaps in the ARS collection coverage. In support of Sub-objective 1C, research on the threat of gene-flow for the genetically engineered glyphosate herbicide resistance trait in alfalfa to the field regeneration site continued and is expected to increase as a second genetically engineered trait in alfalfa has been deregulated (low-lignin). In addition, commercial transgenic alfalfa plantings in the area continue to increase. To monitor this potential gene-flow a total of 14 sentinel plots were included in the regeneration field in 2018. These plots are located on each of the regeneration field corners as well as centrally and are of the ‘Vernal’ alfalfa variety. In 2018, sentinel plots were both covered and uncovered, to assess the potential gene-flow to the field site (uncovered) and into the cages (covered). All sentinel plots are regenerated using standard regeneration protocols. Seed harvested from these plots is destructively sampled/tested for adventitious presence of glyphosate resistance at the end of the season. In addition, conventional commercial alfalfa seed lots being used to establish new hay fields on the Washington State University (WSU) experimental farms are also being tested to establish baseline adventitious presence levels and avoid any high-level transgene contaminated seed lots. From the seven covered ‘Vernal’ alfalfa variety sentinel plots around regeneration fields which were tested in 2018, more than 25,000 seed were tested, and no adventitious presence (AP) was detected. Five out of the seven uncovered plots showed positive results in testing, indicating gene-flow to the regeneration site. These efforts relate to the continued monitoring for adventitious presence of transgenes in the germplasm being regenerated at the Prosser, WA worksite. Monitoring genetically engineered (GE) geneflow and AP in alfalfa is part of Sub-objective 1C. Substantial progress was made on Sub-objective 2A, which focuses on fungal disease resistance. A U.S. Alfalfa Farmer Initiative (USAFRI) which funded a project entitled “Spring black stem and Stemphylium leaf spot resistance screening in the USDA-ARS National Plant Germplasm System’s Medicago spp. genetic resources” was initiated in FY19. Spring black stem (SBS) and Stemphylium leaf spot (SLS), caused by Phoma medicaginis and Stemphylium spp., respectively, are important alfalfa foliar fungal plant pathogens for which good disease resistance is not available in commercial cultivars. The project aims to 1) optimize inoculation protocols for fungal species causing SBS and SLS diseases of alfalfa; 2) systematically screen through alfalfa germplasm for disease resistance to SBS and SLS; 3) define host range for SBS and SLS diseases in subsets of representative Medicago spp. taxa; 4) Evaluate Standard Check variety and inventories for reaction to SBS and SLS diseases; and 5) make data, and associated information, publicly available through presentations, publications and through the USDA-ARS National Plant Germplasm System’s GRIN-Global database. A Washington State University Research Intern position was established and is being supported by this grant. Fungal disease resistance screening in alfalfa is part of Sub-objective 2A. Progress on Sub-objective 2B included efforts to systematically characterize an annual medic (Medicago spp.) genetic resources collection originating from the Crimean Peninsula of Ukraine. The collection consists of close to 100 accession in ten different taxa. These accessions, along with another group of close to 100 reference accessions in the same taxa from the Western Regional Plant Introduction Station's (WRPIS) existing collections, were field established from greenhouse transplants. The plants in the field were used to characterize individual accessions for phenotypic diversity using a set of over 20 individual International Plant Genetic Resources Institute (IPGRI)-generated descriptors. Data is being summarized from the first year and will be combined with second-year data to finalize collecting all descriptors. Prior to field establishment, DNA was extracted for all Crimean and reference accessions and these are being genotyped by collaborators in Stoneville, Mississippi. Comparisons between field generated phenotypic information with genotypic data will aid in determining gaps or redundancies in genetic diversity coverage as well as refine taxonomic relationships in accessions. In support of Sub-objective 2B, a collaborative effort with several research institutions including Arizona State University (alfalfa and relatives), the University of Puerto Rico (trefoil and relatives) and Loyola University of Chicago (clover and relatives) continues. Collaborators at each of these institutions are working towards characterization of temperate adapted forage legume accessions using flow cytometry (ploidy determination) and sequencing of deoxyribonucleic acid (DNA) barcodes. Ploidy determination or estimating number of chromosomes and DNA barcoding are approaches used to voucher or correctly identify germplasm taxa and/or accession inventories. This is especially useful where identities are unknown or where misidentification or mislabeling might have occurred. Accession that were selected and mailed to project collaborators have been DNA-extracted and sequenced. Sequence data is being analyzed and will aid in proper identification for several accessions for which taxa is unknown, in identifying possible mislabeling or misidentification errors in the accessions sequenced as well as in developing and depositing into GenBank voucher DNA barcodes. Possibility exists to summarize these efforts into peer-reviewed publications. In support of Objective 3, a Manual of Operating Procedures (MOP) exists that documents the daily processes followed in implementing the current project. This MOP outlines the best management practices to follow and is continually updated to include new approaches undertaken to execute effectively and efficiently the temperature adapted-forage legume (TFL) germplasm project. The manual has recently been updated to include a few new processes adopted by the project which include 1) the use of both covered and uncovered sentinel plots for monitoring gene flow and adventitious presence of genetically engineered traits to the alfalfa field regeneration site; and 2) references to both the “ARS Procedures and Best Management Practices (BMPs) for Genetically Engineered (GE) 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 documents provide detailed information and guidance on how the National Plant Germplasm System (NPGS) will meet its mission while continuing to implement new techniques and strategies to ensure genetic integrity of the collections in the presence of growing GE traits in agricultural crops.