Research Project: Conservation, Characterization, Evaluation, and Distribution of Grain, Oilseed, Vegetable, Subtropical and Tropical Legume, and Warm Season Grass Genetic Resources and Associated Information

Location: Plant Genetic Resources Conservation Unit

2022 Annual Report

Objectives
1. Efficiently and effectively acquire, distribute, and maintain the safety, genetic integrity, health, and viability of priority grain, oilseed, vegetable, subtropical and tropical legume, and warm season grass genetic resources and associated information. 1.A. Acquire genetic resources to expand the diversity of priority crops and crop wild relatives (CWR) available from the genebank via collection, exchange or other appropriate means. 1.B. Conserve and maintain over 94,000 accessions of priority genetic resources and their associated information, periodically assess these priority genetic resources for viability, trueness to type, and health, and distribute accessions upon request. 1.C. Conduct field and greenhouse regenerations of priority crops and CWR to replenish and safeguard high quality genetic resources in state-of-the-art genebank. 2. Develop more effective genetic resource maintenance, evaluation, or characterization methods and apply them to priority grain, oilseed, vegetable, subtropical and tropical legume, and warm season grass genetic resources. Record and disseminate evaluation and characterization data via GRIN-Global and other data sources. 2.A. Using phenotypic descriptors, evaluate priority crops and CWR for agronomic and horticultural traits and incorporate this data into GRIN-Global. 2.B. Develop and apply nuclear magnetic resonance (NMR), rapid N exceed [nitrogen/protein] analyzer (RNEA), high performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC/MS) procedures to evaluate variation in oil, protein, sugar content, amino acid composition, fatty acid composition, flavonoids, flavors, and other key phytochemicals in priority crops and CWR and incorporate this data into GRIN-Global. 2.C. Develop and apply DNA markers to assess phylogenetic relationships, genetic diversity, population structure, and association with phenotypic traits of priority crops and CWR. Enter DNA genetic marker characterization data into GRIN-Global or other databases (such as GenBank). 3. With other NPGS genebanks and Crop Germplasm Committees, develop, update, document, and implement best management practices and Crop Vulnerability Statements for priority grain, oilseed, vegetable, subtropical and tropical legume, and warm season grass genetic resource and information management.

Approach
Curators will acquire plant genetic resources from collection trips, donations, and exchanges with other gene banks and state universities to adequately conserve the range of crop genetic diversity. Seed from each accession maintained in the collection will be preserved in cold storage to optimize long-term seed viability and reduce the frequency of regeneration. Efforts will continue to conduct standard germination tests on the entire range of crop and crop wild relative accessions in the germplasm collection with emphasis on testing new material and retesting select inventories at ten year intervals. Plant genetic resources (seeds, in-vitro cultures, plants, cuttings, corms, and rhizomes) and associated information will be sent to users worldwide in response to requests received by email, internet, phone, and U.S. mail. Accessions with low seed viability, low seed numbers, original seed only, and age of seed will be targeted for regeneration. Curators will observe and collect phenotypic data using descriptors for each of the accessions/crops grown for regeneration or evaluation. Additional descriptors on classification, local adaptability, and other traits of agricultural importance will be recorded as opportunity permits. Valuable biochemical traits such as oil/fatty acid and protein/amino acid content in oil seed crops; flavonoids and anthocyanins in legumes; flavor and resveratrol in peanuts; protein content in Vigna; protein and mineral content in pearl millet seeds; and fruit color and flavor components in pepper (Capsicum spp.) will be collected, analyzed and made available on the Germplasm Resources Information Network (GRIN-Global). Genetic characterization and evaluation of plant germplasm will be conducted. For genetic characterization of little bluestem, sweet potato and pepper, previously published simple sequence repeat (SSR) markers are available and will be utilized as the focus of the research is not on marker development but rather characterization. For peanut and sorghum, where advanced genomic tools are available, single nucleotide polymorphism (SNP) markers will be used for characterization, association analysis, and design of functional DNA markers. Curators will consult with Crop Germplasm Committees (CGCs) to develop, update, document, and implement Best Management Practices (BMPs) and Crop Vulnerability Statements (CVS) for crops conserved in the genebank. All data including passport, regeneration, and characterization data will be submitted electronically to the Information Technology Specialist or Seed Storage Manager and their designated staff for local storage and uploading to the GRIN-Global database.

Progress Report
A large and highly diverse set of plant germplasm was preserved and distributed to scientists, educators, and plant breeders. A total of 103,176 accessions of 1,602 plant species representing 281 genera were maintained in the Griffin plant genetic resources collection. Over 87% of these accessions were available for distribution to users and over 94% were backed up securely at a second location. A total of 37,230 seed and clonal accessions were distributed upon request to scientists and educators worldwide this fiscal year as of July 13, 2022. Sorghum, cowpea, watermelon, and pepper were the most distributed crops. Clonal collections were continually maintained and distributed to stakeholders. Clonal collections include warm-season grasses, bamboo, Chinese water chestnut, perennial peanut, and sweet potato. Preservation methods include tissue culture, field plots, greenhouse plants, and hydroponics. A total of 2,590 accessions were sent to curators for regeneration. Collaborator regenerations led to successful regeneration of many crops including wild peanut (Griffin, Georgia), sorghum and millet (USDA-ARS, Puerto Rico) and vegetable crops (USDA-ARS, Parlier, California; Rijk Zwaan; Vilmoran; HM Clause; Curry Seed and Chile Company). These activities ensure that the crop genetic resources at the Griffin location are safeguarded for future use to develop new cultivars and identify novel traits and uses in our food and fiber crops. The collaborative project with researchers at the University of Georgia-Tifton Campus to develop molecular diagnostic tools for the detection of peanut clump virus and the Indian peanut clump virus with funding support from the National Plant Disease Recovery System (NPDRS) was completed. Molecular diagnostic assays were developed and standardized for the detection of these two viruses. Protocols were shared with USDA-Animal Plant Health Inspection Service (APHIS) and the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) for validations. These diagnostic tools are needed to screen new peanut germplasm entering the country. With partial support from the National Peanut Board, the collaborative research project with ARS researchers in Stoneville, Mississippi, and the Hudson Alpha Institute, Huntsville, Alabama, to analyze the wild peanut genome was initiated. The sequencing of three wild peanut species has been completed. Increased knowledge of the wild peanut genome will enable peanut breeders to more efficiently cross wild peanut with cultivated peanut. This allows new peanut varieties to be developed with desirable traits originally found in the wild peanut. CRISPR gene editing is a new technology to precisely modify traits for crop improvement. Collaboration continued with Tuskegee University on a gene editing project for peanut with hopes of increasing the health beneficial oleic acid content. In collaboration with ARS researchers in Tifton, Georgia, fatty acid composition was determined in 349 recombinant inbred lines (RIL) from the peanut MAGIC population. This population has parents with high oleic acid content and resistance to both leaf spot and the tomato spotted wilt virus. The goal of the project is to develop peanut germplasm lines with both the high oleic acid trait and disease resistance. In collaboration with the University of Georgia wild peanut lab, three elevated oleic acid lines were identified in populations developed from a synthetic tetraploid peanut. This facilitates the introduction of traits from wild peanut species into cultivated peanut for the development of improved peanut varieties. With the same research group, 100 wild peanut accessions were genotyped with a single nucleotide polymorphism (SNP) array. The collected seeds will be assayed for seed quality traits including oil and protein content, fatty acid composition, and resveratrol content. The purpose is to associate nutritional quality traits with the SNP markers for use in marker-assisted breeding. Collaboration continues with ARS researchers in Charleston, South Carolina, to cross the wild species related to watermelon, Citrullus ecirrhosus, with cultivated watermelon species. This species is being evaluated for resistance to root knot nematodes which are a problem in watermelon production areas in the southeastern United States. A new source of resistance could be used to develop improved watermelon varieties. A collaborative study was initiated with the University of Houston Medical Center and the University of Georgia to determine the genomic basis for sex determination in watermelon. Understanding how chromosome evolution in the genus occurred can be used to better understand the evolution of agriculturally important genes. Pepper accessions were screened for the presence of Tomato Brown Rugose Fruit Virus. The presence of viruses or the lack of knowledge concerning the presence or absence of viruses in germplasm hinders the ability to distribute germplasm and can result in the unintentional distribution of plant pathogens. Collaborative efforts with ARS researchers in Stoneville, Mississippi, to identify and characterize novel uses of bioactive compounds in pepper including capsinoids continued. Capsinoids, the substance that gives peppers their hot pungent flavor, has been shown to be an important nutraceutical compound with many potential health benefits. Glactomannan (gum) concentration was measured in twelve guar accessions using High Performance Liquid Chromatography. Due to its high viscosity, guar gum is a popular polymer used in oil well drilling for hydraulic fracturing and in the food industry as a stabilizer for frozen and baked foods and a thickener for salad dressing. Identification of guar accessions high in gum concentration is useful in the development of guar varieties with improved commercial traits.

Accomplishments
1. Nutritional compounds in blackeye peas. Blackeye peas are used as a vegetable worldwide. However, little is known regarding the flavonol and anthocyanidin variation in seed accessions within the USDA, ARS germplasm collection. Flavonols (quercetin and myricetin) as well as the anthocyanidins (cyanidin, delphinidin) have potential to have antioxidant, anti-cancer, and antimicrobial activities. ARS researchers in Griffin, Georgia, evaluated the qualities of these nutritional components in 38 blackeye pea accessions. Significant variability for quercetin, myricetin, cycanidin, and delphinidin were identified from these accessions. Four accessions were discovered to produce significantly higher concentrations of these phytochemicals. Genetic parameters also showed that improving these phytochemicals is possible through selection. These results can be used by scientists to develop black eye pea varieties with high flavonol and anthocyanidin concentrations.

2. Better understanding the watermelon. Watermelon is an important crop in the US, and abroad. As a crop, it is subject to attack by many diseases, pests, and environmental variables such as drought. Fortunately, there are wild relatives of watermelon that are known to possess traits (genes) that might be used to overcome some of these challenges to production. ARS scientists, in collaboration with colleagues, have sequenced the DNA of Citrullus naudinianus, a desert-dwelling species that is the ancestor of today’s cultivated watermelon. Comparisons of the DNA sequence of Citrullus naudinianus with the DNA sequences of other wild and cultivated watermelons helps to understand how useful traits such as disease, insect or drought resistance, first developed, changed over time, or were lost. This knowledge, in turn, provides the basis for modifying the genetic makeup of the cultivated watermelon so that it contains the useful traits found in its wild relatives, thus facilitating more efficient and secure crop production.

3. Distributing healthy seed. The USDA genebanks distribute seed to the research community. Many pathogens, notably viruses and viroids, can be transmitted on or in seed. The presence of pathogens such as viruses or viroids on or in the seed can limit the ability of the genebank to distribute seed as the presence of certain seed-borne pathogens results in an inability to meet government-regulated seed health requirements. Seed testing is required to determine if certain seed-borne pathogens are present. ARS scientists and collaborators tested approximately 1,000 genebank accessions of pepper and 850 accessions of eggplant for important viruses and viroids. Infected seed inventories and specific pathogens were identified. The information gained has been used to isolate infected seed inventories and to modify genebank seed regeneration and distribution activities. These actions serve to eliminate the unintentional distribution of pathogen-infected seed and the danger(s) to crop production and research that might result from the distribution of infected seed.

4. Extracting compounds with medicinal properties from pepper. Peppers and other fruit are sources of compounds that have value for medicinal purposes or in various commercial applications, if available in sufficient quantity. Capsinoids are a group of compounds unique to pepper that are related to capsaicinoids (the ‘heat’ in hot pepper). Capsinoids have unique medicinal properties and possibly potential as a food additive. Unfortunately, most capsinoids must be synthesized from precursor compounds using a complex and costly procedure. ARS researchers developed a procedure for the large-scale extraction/purification of capsinoids from fresh or dried pepper fruit. To date, this is the only documented procedure for the large-scale isolation/purification of capsinoids from fresh fruit. The material produced via this extraction/purification procedure will facilitate the further evaluation of capsinoids for their medicinal and other uses.

5. Regeneration of vegetable crops germplasm. For plant genetic resources to be effectively utilized, they must first be available to the scientific community. Several crops maintained by ARS researchers in Griffin, Georgia, are difficult to regenerate including okra, peppers, gourds, and watermelon. There is a critical need to regenerate this germplasm. Numerous accessions of these crops were regenerated in the field or greenhouse in Griffin, Georgia, and in collaboration with various private sector enterprises. Trait data was also collected during the regeneration process and uploaded to the Germplasm Resources Information Network (GRIN-Global) for public access. The newly harvested seed and associated information will serve as a basis and stimulus for the utilization of genetic resources for these crops.

6. Peanut germplasm diversity. Germplasm collections provide the raw material to improve commercial crops. To use these resources effectively, information on traits should be available to breeders and other researchers. In the first comprehensive study of the USDA peanut collection, ARS researchers in Griffin, Georgia, measured seed size, oil content and composition, and color traits for all available accessions. Wide ranges in variation for these traits were identified. This is significant because these differences can affect crop yield and nutritional quality. For example, accessions with larger seeds and/or higher oil can significantly increase total yield. Likewise, peanuts higher in oleic acid, which is a component of the oil, are healthier to consume. These data will be a vital reference for selecting peanut breeding materials.

Review Publications
Patel, J.D., Wang, M.L., Dang, P.M., Butts, C.L., Lamb, M.C., Chen, C.Y. 2022. Insights into the genomic architecture of seed and pod quality traits in the U.S. peanut mini-core diversity panel. Plants. 11(7):837. https://doi.org/10.3390/plants11070837.
Morris, J.B. 2022. Multivariate analysis of butterfly pea (Clitoria ternatea L.) genotypes with potentially healthy nutraceuticals and uses. Journal of Dietary Supplement. https://doi.org/10.1080/19390211.2021.2022821.
Mariod, A., Jarret, R.L. 2022. Antioxidant, antimicrobial and antidiabetic activities of Citrullus colocynthis seed oil. In: Mariod, A., editor. Multiple Biological Activities of Unconventional Seed Oils. London, UK: Academic Press. p. 139-146. https://doi.org/10.1016/B978-0-12-824135-6.00005-2.
Cantrell, C.L., Jarret, R.L. 2022. Bulk process for enrichment of capsinoids from capsicum fruit. Processes. 10(2):305. https://doi.org/10.3390/pr10020305.
Munoz-Rodriguez, P., Wells, T., Wood, J., Carruthers, T., Anglin, N.L., Jarret, R.L., Scotland, R. 2022. Discovery and characterisation of sweetpotato’s closest tetraploid relative. New Phytologist. 234:1185-1194. https://doi.org/10.1111/nph.17991.
Morris, J.B., Tonnis, B.D., Wang, M.L., Bhattari, U. 2022. Genetic diversity for quercetin, myricetin, cyanidin, and delphinidin concentrations in 38 blackeye pea (Vigna unguiculata L. Walp.) genotypes for potential use as a functional health vegetable. Journal of Dietary Supplement. https://doi.org/10.1080/19390211.2022.2077881.
Meagher Jr, R.L., Nagoshi, R.N., Fleischer, S.J., Westbrook, J.K., Wright, D.L., Morris, J.B., Brown, J.T., Rowley, A.L. 2022. Areawide management of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), using selected cover crop plants. CABI Agriculture and Bioscience (CABI A&B). 3:1. https://doi.org/10.1186/s43170-021-00069-0.
Massa, A.N., Arias De Ares, R.S., Sorensen, R.B., Sobolev, V., Tallury, S.P., Stalker, T.S., Lamb, M.C. 2021. Evaluation of leaf spot resistance in wild arachis species of section arachis. Peanut Science. 48(2):68-75. https://doi.org/10.3146/PS20-25.1.
Wang, M.L., Wang, H., Zhao, C., Tonnis, B.D., Tallury, S.P., Wang, X., Clevenger, J., Guo, B. 2021. Identification of QTLs for seed dormancy in cultivated peanut using a recombinant inbred line mapping population. Plant Molecular Biology Reporter. https://doi.org/10.1007/s11105-021-01315-5.