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

Location: Plant Germplasm Introduction and Testing Research

2018 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 project which began in January of 2018 continues research from 2090-21000-025-00D, “Temperate Forage Legume Genetic Resource Management, Characterization, and Evaluation”. Please see the report of the previous project for additional information. Progress is being made towards all three objectives and their sub-objectives, all of which fall under National Program 301, Plant Genetic Resources, Genomics, and Genetics Improvement. This project focuses on Problem Statement 1A: Efficiently and Effectively Manage Plant and Microbial Genetic Resources. Introduced plant genetic resources are critical for improving the current crops and developing new crops. For the 2017 calendar year a total of 4,472 accessions (6,743 items) of Medicago (alfalfa and its relatives), Trifolium (clover and its relatives), and Lotus (trefoil and its relatives) spp. were distributed in the form of seed in 178 orders to 160 cooperators. The seed was distributed to 36 U.S. states and to 15 countries internationally. A total of 46 records associated with unique plant collections were updated in GRIN-Global, most of which were 100-seed weight data points. Many digital images were collected; however, none were loaded as needed software in GRIN-Global database was still being developed. During the fiscal year, 13 new acquisitions were incorporated from donations though the Seeds of Success program or through expiring plant variety protection (PVP) protected germplasm from the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. Germinations for viability monitoring was also conducted for 95 accessions. The mostly service-oriented progress reported immediately above relates directly to Objective 1 and Sub-objectives 1A and 1B. As in previous years, sentinel plots of the alfalfa variety ‘Vernal’ have been established around regeneration field site to monitor for pollen movement from commercial genetically engineered alfalfa fields into the regeneration field site. It is important to document and/or quantify possible exposure of non-genetically engineered alfalfa germplasm to genetically engineered traits (that can move in pollen). Located on the periphery of the fields and centrally, sentinel plots are either covered with insect-proof cages or left uncovered. Plots are treated the same as other accessions being regenerated, with cages being installed prior to blooming and only taken down at the time of harvest. Pollinators are included in the cages to aid in seed set. At the end of the season plots are harvested, dried, threshed, seed is cleaned and tested for gene flow and adventitious presence of glyphosate herbicide resistance (i.e., Roundup ready) trait. To date gene flow and adventitious presence has been detected at regeneration field site, but only in uncovered sentinel plots, and only at very low rates. Gene flow monitoring in sentinel plots addresses Objective 1 and Sub-objective 1C. As part of Objective 2, Sub-objective 2B, a panel of approximately 205 annual medic (Medicago spp.) accessions, originally collected in the Crimean region of Ukraine, were selected for a genetic diversity assessment study. Several annual medics are important forage crops (e.g., M. polymorpha - burr clover) and are wild relatives of cultivated alfalfa. DNA has been extracted from three individual plants as well as for one bulk of seven representative plants (10 total plants per accession total) for each of these Crimean accessions. In addition, a subset of four DNA extractions (three individual/one bulk) for approximately 200 select reference taxa accessions for comparison which originate from the proximal regions (mostly countries around the Black Sea) has been included in the study. The DNA will be amplified with a set of molecular (SSR) markers, by collaborators at the USDA-ARS Genomics and Bioinformatics Unit in Stoneville, Mississippi, and resulting data analyzed to estimate genetic diversity, relatedness, redundancies and correct taxonomic identities. The same set of close to 4,000 plants have been field-established for collection of characterization data that will complement the genetic diversity evaluation. 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) is under way. Collaborators at each of these institutions are working towards characterization of temperate adapted forage legume accessions using flow cytometry (ploidy determination) and sequencing of 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. The effort at Arizona State University working on alfalfa and some of its wild relatives (Medicago spp.) was recently completed and data is being summarized for peer-review publication as well as for associating it with specific accessions in the GRIN-Global database. These efforts relate to Objective 2, Sub-objective 2A and 2B, where the collaborative efforts are using molecular techniques to identify diversity/redundancy and where characterization and evaluation data generated will increase useful accession-associated information in GRIN-Global. 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 temperate-adapted forage legume (TFL) germplasm project. The manual has recently been updated to include three new processes adopted by the project which include: 1) the use of sentinel plots for monitoring gene flow and adventitious presence of genetically engineered traits in alfalfa; 2) to use clonal propagation techniques when low numbers of plants (<100) are available for regenerations; and 3) the option of using in vitro/laboratory germination for low-seed number inventories when establishing plants in greenhouses for eventual field regenerations. Updating best management practices is part of Objective 3. A very well attended Alfalfa Crop Germplasm Committee (CGC) meeting was held concomitantly with the bi-annual meeting of the North American Alfalfa Improvement Conference (NAAIC) in June of 2018 in Logan, Utah. By way of an oral report, all participants were informed and updated on the status of the TFL collection. Many of the attendees agreed to actively participate in addressing issues with Alfalfa Standard Tests and Standard Check varieties. Standard Tests and Standard Check varieties in those tests are used for comparison in evaluations of promising germplasm set to be released commercially. The Alfalfa Standard Check varieties are distributed through the TFL project. The renewed activity in the alfalfa CGC is promising and the list of participants will be queried for information when developing the new Crop Vulnerability Statement for Alfalfa. This described work is associated with Objective 3. The ARS lead scientist on this project participated and presented research at the Crop Science Society of America and the Clover and Special Purpose Legume Crop Germplasm Committee meetings held in Tampa, Florida, as well as at the North American Alfalfa Improvement Conference and Alfalfa Crop Germplasm Committee meetings held in Logan, Utah. In addition, he participated and presented research at the biannual meeting of the Plant Germplasm Operations Committee Conference (PGOC) Curator Workshop in Beltsville, Maryland, and has been elected to serve as Chair-elect of the PGOC.