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

Location: National Clonal Germplasm Repository

2019 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, Corvallis, Oregon, is a genebank that conserves temperate fruits, nuts, and specialty crops. The genebank conserves more than 12,000 accessions of 30 genera of horticultural and agronomic crops. It is the repository for hazelnuts, strawberries, hops, mint, pears, currants, gooseberries, blackberries, raspberries, blueberries, cranberries and their crop wild relatives. The primary collections are like a library of living plants, maintained as orchards in the field, or containerized plants in the screenhouse, or seeds representing species populations. Seeds are preserved in freezers to extend their viability. Alternative secondary storage is maintained through replicate plantings in remote locations at Parlier, California, and in vitro cultures and cryogenic preservation at a collaborating site, Fort Collins, Colorado. In addition, back-up collections of kiwifruit and butternuts are planted in Corvallis for the Davis, California genebank. More than 7,221 accessions were shipped during the year to researchers throughout the world. The ARS researchers worked with the requestors and quarantine inspectors to ensure that the plant materials met importation permit requirements and had USDA phytosanitary certification, when required. The molecular genetics laboratory at the genebank developed DNA fingerprinting sets for many 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 being entered into the Germplasm Resources Information Network database for public accessibility. More than 440 visitors toured the repository this past year. ARS researchers collaborated with about 75 international scientists during this past year and obtained extramural funding for evaluation of assigned genera. Collaborations included competitive grant opportunities in Specialty Crop Research Initiatives, commodity commission funding such as from Washington and California Tree fruit commissions, North American Bramble Growers, ARS Plant Evaluation Grants, and USDA Animal and Plant Health Inspection Service.

Accomplishments
1. Black currant reversion virus (BRV) in black currant germplasm at the National Clonal Germplasm Repository (NCGR). ARS scientists confirmed the presence of black currant reversion virus (BCR) in black currant germplasm accessions in Corvallis, Oregon. Total RNA was extracted from two infected black currant accessions and sequenced amplicons matched the virus genome with >88% identity. A note was published concerning the presence of the virus. The entire USDA black currant germplasm collection was sampled in April, July and October 2018 and tested negative by two different polymerase chain reaction techniques. New primers will be designed using virus sequence information so that the distribution of BRV in Ribes nigrum germplasm can be assessed. This virus is of quarantine significance to any black currant imported into the U.S.

2. Sequencing of 2,000 pears at the National Clonal Germplasm Repository at Corvallis, Oregon. ARS scientists sequenced the DNA from more than 2,000 pear genotypes from the USDA pear genebank in Corvallis, Oregon. A highly efficient array was developed for genetic analysis, evaluation for high-density mapping and characterization of these pears. This data will allow future research to link the phenotype, such as fireblight resistant or cold tolerant, with the genotype. Commercial pear production is concentrated in the Northwestern U.S. and was valued at $519 million in 2017.

3. Variable salinity responses of strawberries. ARS scientists in Corvallis, Oregon, examined the variable salinity responses and comparative gene expression in alpine strawberries. The expression of 11 genes involved in sodium and chloride ion transport revealed differences in traits responsible for salt tolerance. Sodium sequestration in vacuoles and the chloride channel gene may play an important role in salt tolerance. This determination in diploid strawberries will need to be examined for the hybrid octoploid strawberry of commerce. California and Florida are the top two strawberry producing states in the U.S. and both have salinity issues regarding strawberry production. In 2017 the U.S. produced 1.6 billion pounds of strawberries valued at $3.5 billion.

4. Survey of mint crop ancestors. ARS scientists in Corvallis, Oregon, surveyed the genetic diversity of peppermint and spearmint crop ancestors and determined their ploidy, essential oil composition, and relative Verticillium wilt resistance. This study provided updates of accession descriptions in the Germplasm Research Information Network database. The study will be used to increase the utility of the Mentha collection to the research community. In 2018, the farm gate value of U.S.-produced peppermint was $106 million.

5. Hazelnut DNA fingerprinting. ARS scientists in Corvallis, Oregon, developed a reliable hazelnut reference database of 195 accessions through the implementation of a DNA fingerprinting set. Implementation of this test together with the addition of more unique accessions to the reference database, will help verification of trueness-to-type of economically important cultivars which is of critical interest to nurserymen and growers in the hazelnut industry. In 2016, the world produced 44,000 tons of hazelnuts valued at $118 million, with Turkey as the number 1 producing country. Recent weather and disease issues in Turkey have increased the demand from the U.S. Oregon, with crop value of $73 million in 2017, produces about 98% of the U.S. hazelnut crop, and Oregon's hazelnuts are bigger and more flavorful than the Turkish nuts.

6. Black raspberry genome sequence. ARS scientists in Corvallis, Oregon, established a reference chromosome-scale genome sequence for black raspberry using new techniques. This updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rose family. The genome sequence will enable the development of marker assisted breeding in these berry crops. Black raspberries are an American crop that is produced in California, Oregon, and Washington, with a value for fresh and processed product from the three states valued at $516 million in 2017.

7. Identity of heritage blackberry cultivars. ARS scientists in Corvallis, Oregon, developed a concise fingerprinting set of simple sequence markers to confirm the identity of blackberry cultivars. This technique enabled the scientists to recognize the original genotype of 'Boysen' as well as determine imposters that were being sold in the nursery trade. This technique allowed the scientists to compare DNA samples of parents, offspring, and the cultivars to determine trueness-to-type. This technique can now be used by commercial laboratories to resolve grower identity issues. In 2017, the U.S. blackberry production was valued at $31.1 million and is increasing.

8. Domestication of the strawberry: alleles from distinct ancestral genomes. ARS scientists in Corvallis, Oregon, examined the genome of domestic strawberries of commerce to determine polyploid genome complexity resulting from migration, selection, and population bottlenecks. Profiles of 1,300 octoploid strawberries from throughout history were examined. The globally important California population adapted to sub-tropical coastal habitats diverged from types selected from eastern North America and Europe. Other temperate octoploids selected elsewhere in North America were chosen from the photoperiod-insensitive Virginian strawberry and the beach strawberry. Diverse new strawberry cultivars will continue to enable increased productivity on existing California acreage and elsewhere so that the growers and consumers benefit as seen in strawberry production valued at $5.17 billion in 2017.

Review Publications
Hardigan, M., Poortan, T., Acharya, C., Cole, G., Hummer, K.E., Bassil, N.V., Edger, P., Knapp, S.J. 2018. Domestication of temperate and coastal hybrids with distinct ancestral gene selection in octoploid strawberry. The Plant Genome. 11(3):180049. https://doi.org/10.3835/plantgenome2018.07.0049.
Zurn, J.D., Zlesak, D., Holen, M., Bradeen, J., Hokanson, S., Bassil, N.V. 2018. Mapping a novel black spot resistance locus in the climbing rose Brite Eyes™ (‘RADbrite’). Frontiers in Plant Science. 9:1730. https://doi.org/10.3389/fpls.2018.01730.
Verma, S., Evans, K., Guan, Y., Luby, J., Rosyara, U., Howard, N., Bassil, N.V., Bink, M., Van De Weg, E., Peace, C. 2019. Two large-effect QTLs, Ma and Ma3, determine genetic potential for acidity in apple fruit: Breeding insights from a multi-family study. Tree Genetics and Genomes. 15:18. https://doi.org/10.1007/s11295-019-1324-y.
Hummer, K.E., Williams, K.A., Bushakra, J. 2019. North American crop wild relatives of temperate berries (Fragaria L., Ribes L., Rubus L., and Vaccinium L.). In: Greene S., Williams K., Khoury C., Kantar M., Marek L., editors. North American Crop Wild Relatives, Volume 2. Cham, Switzerland: Springer. p. 283-327. https://doi.org/10.1007/978-3-319-97121-6_9.
Vining, K., Pandelova, I., Hummer, K.E., Bassil, N.V., Contreras, R., Neill, K., Chen, H., Parrish, A., Lange, B.M. 2019. Genetic diversity survey of Mentha aquatica L. and Mentha suaveolens Ehrh., mint crop ancestors. Genetic Resources and Crop Evolution. 66:825-845. https://doi.org/10.1007/s10722-019-00750-4.
Mccoy, J., Young, J.H., Nifong, J.M., Hummer, K.E., De Noma, J.S., Avendano-Arrazate, C.H., Greene, S.L., Kantar, M.B. 2019. Species for medicinal and social use with an emphasis on Theobroma cacao L. (cacao), Nicotiana tabacum L. (tobacco), Actaea racemosa L. (black cohosh), and Humulus lupulus L. (hops). In: Greene S., Williams K., Khoury C., Kantar M., Marek L., editors. North American Crop Wild Relatives, Volume 2. Cham, Switzerland: Springer. p. 645-692. https://doi.org/10.1007/978-3-319-97121-6_19.
Carter, K., Zurn, J.D., Bassil, N.V., Finn, C.E., Hummer, K.E. 2019. The importance of being 'Boysen': Examining genotypic variation with simple sequence repeat markers. Journal of American Pomological Society. 73:47-52.
Sandhu, D., Pudussery, M.V., Ferreira, J.F., Liu, X., Pallete, A., Grover, K.K., Hummer, K.E. 2019. Variable salinity responses and comparative gene expression in woodland strawberry genotypes. Scientia Horticulturae. 254:61-69. https://doi.org/10.1016/j.scienta.2019.04.071.
Schlautman, B., Zalapa, J.E., Covarrubias-Pazaran, G., Rodriguez-Bonilla, L., Hummer, K.E., Bassil, N.V., Smith, T. 2018. Genetic diversity and cultivar variants in the NCGR cranberry (Vaccinium macrocarpon Aiton) collection. Journal of Genetics. 97(5):1339–1351. https://doi.org/10.1007/s12041-018-1036-3.
Bassil, N.V. 2019. David Grandison Fairchild: Plant hunter extraordinaire and father of foreign plant introduction. Journal of American Pomological Society. 73:139-144.
Volk, G.M., Henk, A.D., Richards, C.M., Bassil, N.V., Postman, J.D. 2018. Chloroplast sequence data differentiate Maleae, and specifically Pyrus, species in the USDA-ARS National Plant Germplasm System. Genetic Resources and Crop Evolution. 66(1):5-15. https://doi.org/10.1007/s10722-018-0691-9.
Zurn, J.D., Ho, T., Li, R., Bassil, N.V., Tzanetakis, I., Martin, R.R., Postman, J.D. 2019. First report of Blackcurrant reversion virus in Ribes nigrum germplasm in the United States. Plant Disease. 103:1051. https://doi.org/10.1094/PDIS-03-18-0526-PDN.
Montanari, S., Bianco, L., Allen, B., Martinez-Garcia, P., Bassil, N.V., Postman, J.D., Chagne, D., Evans, K., Dhingra, A., Troggio, M., Neale, D. 2019. Development of a highly efficient Axiom® 70K SNP array for Pyrus and evaluation for high-density mapping and germplasm. BMC Genomics. 20:331. https://doi.org/10.1186/s12864-019-5712-3.
Finn, C.E., Strik, B., Mackey, T.A., Jones, P., Bassil, N.V., Martin, R.R. 2019. ‘Echo’ ornamental reflowering blueberry. HortScience. 54(2):368–370. https://doi.org/10.21273/HORTSCI13646-18.
Finn, C.E., Strik, B., Yorgey, B., Peterson, M.E., Jones, P., Lee, J., Bassil, N.V., Martin, R.R. 2019. ‘Hall’s Beauty’ thornless trailing blackberry. HortScience. 54(2):371-376. https://doi.org/10.21273/HORTSCI13678-18.