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

Location: National Clonal Germplasm Repository

2023 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
This is the final report for project 2072-21000-049-000D, Management of Temperate-Adapted Fruit, Nut, and Specialty Crop Genetic Resources and Associated Information, which has been replaced by new project 2072-21000-059-000D, Conservation and Utilization of Temperate-Adapted Fruit, Nut, and Other Specialty Crop Genetic Resources. For additional information, see the new project 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 12,700 unique accessions represented by over 26,000 plant and seed specimen 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 on-site and off-site in Fort Collins, Colorado, and Spitsbergen, Norway. Valuable or vulnerable accessions are backed up as dormant buds or shoot apices under cryogenic conditions at a collaborating site in Fort 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, Oregon, for the ARS Davis, California, genebank and of rhubarb for the ARS genebank in Pullman, Washington. In support of Objective 1, two plant exploration trips were taken and included a trip to western Colorado to collect wild North American hop resources and to Virginia to obtain blueberry and other fruit crop wild relatives. These combined trips resulted in an increase of 111 new accessions to our strawberry, blueberry, hop, blackberry, raspberry, currant, and gooseberry collections and 43 plant vouchers from up to 22 taxa to the U.S. National Arboretum, Washington D.C. In addition to these expeditions, we worked with collaborators to obtain over 450 new accessions to add to our collections and ensure threatened plant species are properly conserved. After establishment, plant and seed accessions were preserved at the genebank in Corvallis, Oregon, and more than 31,000 unique plant specimen were distributed in 4,377 orders to stakeholders throughout the world for research purposes. Up to 4,492 images of fruit, plant, and their characteristics, approximately 3,500 genotypic and 18,513 phenotypic observations were made publicly available through the Germplasm Resources Information Network (GRIN) database and can be used by researchers worldwide. In support of Objective 2, ARS researchers in Corvallis, Oregon, identified blueberry cultivars that are potentially resistant to Blueberry shock virus, blueberry species that are resistant to the spotted wing drosophila fruit fly, pear and quince trees that are tolerant to leaf spot, rust, mildew and fireblight, and black currant accessions that are free of Black currant reversion virus. Forty-eight strawberry plants were found to be infected with Strawberry mild yellow edge virus. Virus-free strawberry plants were established using young apex culture. Virus-free plant representatives were also established from 18 virus-infected hop accessions. These virus-free plants replaced the infected plants in the respective collections. We also collaborated with ARS scientists at Fort Collins, Colorado, to establish a cost- and time-effective protocol for back-up of important pear, currant and gooseberry trees/plants through cryopreservation of dormant buds. In support of Objective 3, ARS researchers developed Crop Vulnerability Statements for currant, gooseberries and strawberries and made them publicly available through the Small Fruit Crop Germplasm Committee website. These reports encompass: biological features, ecogeographic distribution of species, genetic basis of crop production, tribal use of primitive forms, economics of domestic and international production, crop vulnerabilities and threats, genetic uniformity, biotechnology of breeding, genetic erosion in situ, current and emerging biotic and abiotic threats, germplasm accessibility, genebank resources, holdings, gaps, designations of primary secondary and tertiary crop wild relatives, acquisition process, distribution and outreach, future goals, and facilities and equipment needed for off-site conservation. In support of Objective 4, the molecular genetics laboratory at the genebank developed DNA fingerprinting sets and used them to establish databases of DNA-based profiles for economically important blueberry, blackberry, hazelnut, hop, pear, and strawberry cultivars in the collection. In addition, we developed and implemented new genotyping techniques to identify or confirm chromosomal locations of fire blight resistance in pear, sweetness in blackberry as well as continuous flowering and charcoal rot resistance in strawberry. DNA-based diagnostic tests were established for crown rot resistance in strawberry and existing DNA tests for strawberry were compiled and documented in a publicly available DNA Testing Handbook for strawberry. Plant and fruit quality characteristics were determined for over 280 cultivars of strawberry and data were entered into the GRIN database for public accessibility. In support of Objectives 1 and 4, ARS scientists in Corvallis, Oregon, collaborated with about 75 international scientists during these past years to enhance conservation of valuable genetic resources.

Accomplishments
1. A more targeted approach for evaluating texture in blueberry. The consumption of blueberries has increased rapidly in the last decade and blueberry production has expanded to new environments. The industry is facing new challenges with consumers demanding better quality fruit year-round. Fruit texture is one of the most critical parameters of fruit quality in blueberry and there is a need for a standard method to assess this quality for developing new blueberry cultivars. ARS researchers in Corvallis, Oregon, and their collaborators evaluated two different methods for assessing fruit texture from plants grown in different environments. Results confirmed the accuracy and discriminative power of texture analysis across cultivars and selections in different locations and quantified how texture is influenced by environmental differences. This information provides breeders with more accurate techniques for plant selections.

2. A new blackberry genomic sequence to assist berry breeders and scientists. Blackberries are the fourth most economically important berry crop worldwide, yet very few genomic resources exist for blackberries. ARS researchers in Corvallis, Oregon, and their collaborators genetically characterized the diploid blackberry germplasm accession ‘Hillquist’, the only known source of primocane-fruiting (annual-fruiting) character used in fresh-market blackberry breeding programs and is used in the pedigree of many important cultivars world-wide. Results identified several possible candidate genes for primocane-fruiting. This information will facilitate future studies in raspberry and blackberry biology and genetics and strengthen applied breeding programs.

Review Publications
Hummer, K.E., Bassil, N.V. 2023. Confirming clonal identity: A case study in blueberries. Acta Horticulturae. 1357:129-136. https://doi.org/10.17660/ActaHortic.2023.1357.19.
Yalcin, O., Bassil, N.V., Kafkas, N.E. 2022. Wild edible small fruits in Turkey and their fruit characteristics. Proceedings of the Latvian Academy of Sciences. 76(4):417-423. https://doi.org/10.2478/prolas-2022-0065.
Bruna, T., Aryal, R., Dudchenko, O., Sargent, D.J., Mead, D., Buti, M., Cavallini, A., Hytonen, T., Andres, J., Pham, M., Weisz, D., Mascagni, F., Usai, G., Natali, L., Bassil, N.V., Fernandez, G.E., Lomsadze, A., Armour, M., Olukolu, B., Poorten, T., Britton, C., Davik, J., Ashrafi, H., Aiden, E.L., Borodovsky, M., Worthington, M. 2022. A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. "Hillquist"). Genes, Genomes, Genetics. 13(2). Article jkac289. https://doi.org/10.1093/g3journal/jkac289.
Reinhold, L.A., Pscheidt, J.W. 2023. Diagnostic and historical surveys of sweet cherry (Prunus avium) virus and virus-like diseases in Oregon. Plant Disease. 107(3):633-643. https://doi.org/10.1094/PDIS-02-21-0327-SR.
Hummer, K., Bassil, N.V., Zurn, J., Amyotte, B. 2022. Phenotypic characterization of a strawberry (Fragaria ×ananassa Duchesne ex Rosier) diversity collection. Plants, People, Planet. 5(2):209-224. https://doi.org/10.1002/ppp3.10316.
Redpath, L., Gumpertz, M., Ballington, J., Bassil, N.V., Ashrafi, H. 2021. Genotype, environment, year, and harvest effects on fruit quality traits of five blueberry (Vaccinium corymbosum L.) cultivars. Agronomy. 11(9). Article 1788. https://doi.org/10.3390/agronomy11091788.
Mengist, M.F., Grace, M.H., Mackey, T.A., Munoz, B., Pucker, B., Bassil, N.V., Luby, C.H., Ferruzzi, M., Lila, M., Lorizzo, M. 2022. Dissecting the genetic basis of bioactive metabolites and fruit quality traits in blueberries (Vaccinium corymbosum L.). Frontiers in Plant Science. 13. Article 964656. https://doi.org/10.3389/fpls.2022.964656.
Giongo, L., Ajelli, M., Pottorff, M., Coe, K., Perkins-Veazie, P., Bassil, N.V., Hummer, K.E., Farneti, B., Iorizzo, M. 2023. Comparative study on texture: A key for blueberry quality breeding. Acta Horticulturae. 1357:107-114. https://doi.org/10.17660/ActaHortic.2023.1357.16.
Mengist, M., Bostan, H., De Paola, D., Teresi, S., Platts, A., Cremona, G., Qi, X., Mackey, T.A., Bassil, N.V., Ashrafi, H., Giongo, L., Jibran, R., Chagne, D., Bianco, L., Lila, M., Rowland, L.J., Iovene, M., Edger, P.P., Iorizzo, M. 2022. Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum). New Phytologist. 237(3):1024–1039. https://doi.org/10.1111/nph.18428.