Project : USDA ARS

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Research Project: Management of Temperate-Adapted Fruit, Nut, and Specialty Crop Genetic Resources and Associated Information

Location: National Clonal Germplasm Repository

2021 Annual Report

Objectives
1: Efficiently & effectively acquire priority temperate-adapted fruit, nut, & other specialty crop genetic resources; maintain their safety, genetic integrity, health & viability; & distribute them & associated information worldwide. 1A Research heritage cultivars to broaden knowledge of the historical cultivar base. Analyze NCGR-Corvallis crop wild relative collections for gaps in taxa, localities, & diversity. 1B Acquire international & domestic germplasm samples of Corylus, Fragaria, Humulus, Pyrus, Mentha, Ribes, Rubus, Vaccinium, & their crop wild relatives via plant exploration & exchange. Fill gaps, & as opportunities arise through country agreements, acquire elite/enhanced breeding lines & heritage cultivars. 1C Efficiently & effectively establish & maintain crop genetic resources emphasizing temperate fruit, nut, & specialty crop germplasm. Identify duplication & eliminate unnecessary redundancy to maximize efficient germplasm management. 1D Distribute germplasm during the appropriate season for each crop to national & international researchers at public & private institutions & to educational groups. 2: Develop more effective genetic resource maintenance, backup, pathogen testing, & pathogen-elimination methods & apply them to priority temperate-adapted fruit, nut, & other specialty crop genetic resources. 2A Develop improved methods for conservation of temperate crop germplasm in field plantings & container production in protected environments; particularly examining pruning, media, irrigation & integrated pest management. 2B Collaborate with scientists at remote locations to improve backup conservation methods for our 8 primary genera, including growing plants in field plantings, tissue culture storage, & long term cryopreservation. 2C Apply bioassay, ELISA, & develop improved RT-PCR tests for important viruses, viroids, & phytoplasmas in primary plant collections. 2D Eliminate pathogens from select accessions with improved methods for heat therapy & meristem culture. 3: With other NPGS genebanks & Crop Germplasm Committees, develop, update, document, & implement best management practices & Crop Vulnerability Statements for priority temperate-adapted fruit, nut, & other specialty crop genetic resource & information management. 4: Develop more effective genetic resource characterization & evaluation methods, & apply them to priority temperate-adapted fruit, nut, & other specialty crop genetic resources. Record & disseminate characterization & evaluation data via GRIN-Global & other data sources. 4A Establish baseline genetic profiles for core collections with existing DNA fingerprinting sets & develop or optimize fingerprinting sets & enter information to databases. 4B Implement new high throughput genotyping systems in genetic diversity assessment (Fragaria, Pyrus), linkage & trait mapping (Rubus, Fragaria & Pyrus) and phylogenetic analysis (Rubus) & enter information to databases. 4C Develop & apply DNA tests that help identify phenotypes or useful horticultural traits for Fragaria, Rubus & Pyrus. 4D Evaluate phenotypes of flowering, ploidy, disease resistances, & upload data & image characterization to GRIN-Global.

Approach
Objective 1: Acquire, maintain, and distribute germplasm The NCGR-Corvallis is responsible for temperate fruit, nut, and specialty crop genera: Corylus, Fragaria, Humulus, Mentha, Pyrus, Ribes, Rubus, and Vaccinium, as primary collections, and Actinidia and Juglans (J. cinerea) as security backup for other genebanks. Limited amounts of crop wild relative reference collections are also preserved. Primary collections are maintained in greenhouses, screenhouses, field, and as seed. Wild species are maintained as seed stored at -18 C, with some living plant representatives. Heritage cultivars and selections are maintained as clones. Gaps in the collections are determined by research using botanical and horticultural references and species analysis software, as well as referrals through scientific colleagues. New accessions are obtained through plant exploration and exchange. When new accessions are received, information is entered to GRIN-Global and the plant is propagated. Identity is checked by morphology. Orders are received through GRIN-Global and processed in the appropriate season for the crop and plant form, according to requestor needs. Phytosanitary certification is obtained and materials are distributed according to international, regional, and local plant importation regulations. Objective 2: Improved maintenance, secure backup Inventory locations are maintained in GRIN-Global. Clones are re-propagated according to crop to maintain vigor. Pathogen status is evaluated and recorded. Core collections are established and maintained in tissue culture (for backup and distribution); protocols for cryopreservation of dormant buds of the woody plants are being established. Backup greenhouse collections are maintained for the pear and pear relative field collections. Alternative backup procedures and remote backup locations are arranged and recorded. Coordination of in situ conservation of native US germplasm is continuing with other agencies. Objective 3: Vulnerability statements and operations manual Are prepared by the curator in collaboration with the Crop Germplasm Committees. Statements for the currants, gooseberries, mint, hops, and pears will be completed. Statements for strawberries, blackberries, raspberries, have been completed and will be annually updated. Statements are reviewed and revised by expert committees. Approved statements are uploaded to GRIN-Global. The operations manual for the unit will be updated. Objective 4: Characterization and evaluation Cultivar identification will be expanded with new markers and sequencing techniques. Identity of genotypes is being compared globally with clones in other genebanks. Phenotypes of accessions are being evaluated and linked with genotypes. Linkage maps and QTL association are being used for the development of marker-based tests for germplasm characterization traits of crops in the NCGR collection.

Progress Report
The USDA ARS National Clonal Germplasm Repository in Corvallis, Oregon, is a genebank that conserves temperate fruits, nuts, and specialty crops for research reference. The genebank continues to conserve more than 12,000 accessions of eight genera of horticultural and agronomic crops along with their wild relatives. This genebank is assigned to conserve hazelnuts, strawberries, hops, mint, pears, currants, gooseberries, blackberries, raspberries, blueberries, and cranberries. The primary collections are a library of living plants, maintained as orchards in the field, containerized plants in the screenhouse, or seeds representing species populations. Seeds are preserved in freezers to extend their viability. Alternative secondary storage is maintained on-site through seed lots, dormant buds, and other cryogenic techniques at a collaborating site in Ft. Collins, Colorado. A subset of the hazelnut collection is planted in collaboration at an ARS site in Parlier, California. In addition, duplicate collections of kiwifruit and butternuts are planted in Corvallis for the ARS Davis, California, genebank. Plant explorations are planned to expand the genebank to obtain: strawberries that have genes for disease resistance and continuous blooming; raspberries and blueberries that are low chilling; and pears and their relatives that are dwarfing, disease resistant, or cold hardy. The site staff works with quarantine inspectors to insure that the plant materials meet importation permit requirements and have USDA phytosanitary certification when required. The genebank distributes plant material to researchers throughout the world. More than 7,200 accessions were shipped during 2020 and 2021 to requestors for research collaboration. The molecular genetics laboratory at the genebank developed DNA fingerprinting sets for economically important cultivars in the collection. In addition, scientists are performing studies to link genotype and phenotype for specific plants. Gender, ploidy, and phenotypes are being determined for the major fruit cultivars of the collection. This data is entered into the Germplasm Resources Information Network (GRIN) database for public accessibility. The unit scientists collaborated with about 75 international scientists during this past year and obtained extramural funding for evaluation of assigned genera. This collaboration was through competitive grant opportunities in Specialty Crop Research Initiatives, commodity commission funding such as from Oregon Blueberry Commission, ARS Crop Germplasm Committee Evaluation Grants, USDA Plant Exploration Grants, and USDA Animal and Plant Health Inspection Service.

Accomplishments
1. Anthocyanins in wild relatives of strawberry (Fragaria L.). ARS scientists in Corvallis, Oregon, worked with researchers at Oregon State University and Plant Resources Center in Hanoi, Vietnam, to determine the total anthocyanin content and profiles in ripe fruit of nine strawberry species and one out-group, Potentilla indica L. Anthocyanin profiles were obtained using high-performance liquid chromatograph (HPLC). Red-fruited samples had varying amounts of typical strawberry anthocyanins but in varying proportions. White-fruited types contained the three compounds in much lower quantities and different proportions. Evaluation of anthocyanins in wild species will inform breeders of fruit quality for broadening crosses to increase the gene pool. In 2017, the United States produced 1.6 billion pounds of strawberries valued at nearly $4.5 billion.

2. Genetic diversity in the cultivated strawberry (Fragaria ×ananassa) collection at the NCGR. ARS scientists in Corvallis, Oregon, genotyped 539 of 560 cultivated strawberry accessions in the national USDA collection. Clustering analysis using 4,033 markers revealed eight sub-populations associated with different geographic breeding centers. Two 100 accession core collections were determined: one represented a uniform distribution of the gene space, and the other its maximum genetic diversity. Pedigree linkages were also confirmed in the collection. Accessions containing disease resistance associated genes for anthracnose fruit rot, Colletotrichum crown rot, charcoal rot, and Phytophthora crown rot were also identified. These new core collections will allow for breeders and researchers to more efficiently utilize the F. ×ananassa collection in developing new cultivars with desired traits.

3. Evaluated genotype x environment interactions for predicting sweetness in strawberry. Strawberry fruit flavor is due to a complex mix of sugars, acids, and aromatic compounds. The majority of strawberry soluble solids are sugars, and soluble solid content (SSC) is used as a proxy to determine sweetness and has been a target for breeders. A strong genotype × environment (G × E) interaction has been observed for SSC, causing difficulties when studying the genetics underlying SSC in individual environments. In this study, ARS scientists in Corvallis, Oregon, collected genotypic and SSC phenotypic data for 2,064 individuals from seven breeding programs (located in the United States, Spain, United Kingdom, and Australia). Population structure analysis of the 2,064 accessions revealed that populations were clustered into two large groups consisting primarily of subtropical or temperate strawberry accessions. Three models for these subpopulation categories were developed and assessed for their ability to improve prediction accuracy in the presence of population structure. Of the approaches investigated, two were found to have the highest prediction accuracy for SSC. The approaches investigated in this study will provide new insights for the practical implementation of genomic selection in strawberry improvement programs.

4. Berberis hypoxantha C.Y. Wu ex S.Y. Bao (Berberidaceae), a new record for Vietnamese flora. ARS scientists from Corvallis, Oregon, collaborated with scientists in Vietnam and the United Kingdom concerning the identification of a unique barberry species (Berberis hypoxantha) in Lao Cai, Vietnam. This shrub was first observed on Fansipan Mountain during a USDA-sponsored plant exploration expedition in 2015. This high elevation evergreen species has yellow flowers and reduced prickles as compared with other species of the genus. Barberry, known as a hardy garden shrub in the United States, has about 500 species distributed throughout the world. Some species are also valued for their edible berries for culinary and traditional medicinal applications. This is the first report for this endemic species in Vietnam.

5. Developed two hop fingerprinting sets and used them to genotype hop collections. ARS scientists from Corvallis, Oregon, developed two fingerprinting sets for hop: a 9-SSR fingerprinting set and a kompetitive allele specific PCR (KASP) assay of 25 single nucleotide polymorphisms (SNPs). The SSR set was used to genotype 629 hop accessions from collections kept by USDA and University of Nebraska-Lincoln. It identified unique genotypes except for 89 sets of synonymous samples. Population structure analysis clustered accessions into wild North American (WNA) and cultivated groups. Parentage and sibship analyses were used to identify true-to-type cultivars. The SSR fingerprinting set distinguished cultivars, selections, and WNA accessions, while the KASP assays were unable to distinguish the WNA samples. Both fingerprinting sets are valuable tools for identity confirmation and parentage analysis in hop.

U.S. DEPARTMENT OF AGRICULTURE

National Clonal Germplasm Repository: Corvallis, OR