Objective 1: Develop new improved small fruit cultivars for the commercial fruit industry, including, thornless, machine harvestable blackberry cultivars with excellent flavor; high yielding, virus tolerant, high quality strawberry cultivars with outstanding processing characteristics; virus tolerant, high yielding, black raspberry selections with flavor and color characteristics similar to the current standard cultivars; and high quality primocane fruiting red raspberry cultivars with broad adaptation. Objective 2: Evaluate, and incorporate new sources of genetic variability from Rubus, Vaccinium, Fragaria and other minor crops. Specifically, selections of Rubus occidentalis populations from across the country will be evaluated for fruit quality, plant growth, and aphid resistance, and strawberry populations for the presence of repeat flowering characteristics. • Subobjective 2A: Incorporate new/novel germplasm into breeding material to enhance adaptation. • Subobjective 2B: Determine whether trait-associated markers can be developed and if developed, can they be used to identify germplasm that expresses a phenotype of interest. Objective 3: Characterize viruses that infect berry crops, and develop management strategies to minimize the impact of virus diseases on these crops. • Subobjective 3A: Sequence novel viruses of small fruit crops and develop assays for their rapid detection, emphasis will be on raspberry leaf curl disease and two novel virus-like diseases of grapevine. • Subobjective 3B: Determine if endogenous Rubus yellow net virus (RYNV) in raspberry genomes pose a risk of long distance virus spread. • Subobjective 3C: Eradicate Blueberry fruit drop associated virus from blueberry fields in the U.S. Objective 4: Develop virus-tested planting stocks of berry cultivars for fruit production using thermal therapy and other virus elimination techniques, and test RNAi therapy as a new method for virus elimination in berry crops.
For each crop, a modified recurrent mass selection system will be used. Individuals that are selected in a given generation will be intercrossed to produce the next generation. For Rubus spp. and strawberry 4000-6000 seedlings, and 2500-4000 blueberry seedlings from 30-100 crosses will be evaluated annually. Approximately 0.5 to 1.0% of the seedlings are selected based on subjectively evaluated fruit quality, plant performance, ripening period, and yield. Frozen samples will be evaluated for processing characteristics. Selections identified as being superior will be propagated for commercial trial and release. For nursery production, the ARS virology program in Corvallis, OR will help produce virus-tested propagation material. To broaden the germplasm base, superior individuals or representatives of superior populations of small fruit species will be crossed among themselves or with advanced selections or cultivars. Selections from these crosses will be used in our breeding program and distributed to other breeders. Emphasis will be on aphid resistance, disease resistance, fruit quality, thornlessness and tolerance to abiotic stresses associated with growing these crops in regions with more diverse climates. This program collaborates with molecular geneticists in efforts to bridge the gap between genomics and applied plant breeding in berry crops. Our program helps to determine the mapping populations to develop or genotypes to include and how the various phenotypic traits (phenological, reproductive, and vegetative) will be evaluated. The phenotyping for each project is being coordinated across multiple locations with different climatic conditions. For raspberry leaf curl disease, suspect raspberry samples will be collected and their virome analyzed using Next Generation Sequencing. This approach will also be used to examine two novel diseases of grapevines in Oregon. Diagnostic assays will be developed and used for epidemiology studies, certification and quarantine purposes. Virus vectors will be identified and strategies for vector control developed as a means to manage virus diseases. Total genomic sequencing of five Rubus cultivars will be used to determine if the inserted RYNV sequences represent full-length or partial RYNV and if all insertions are at the same site. If the RYNV insertions are at the same site in each of these cultivars it would indicate that the insertion may have happened once and been passed on in breeding programs. Several approaches will be tested for applying gene silencing as a tool to eliminate viruses from growing points of plants (meristematic dome and several leaf primordia): 1. Virus specific dsRNA will be produced and provided to plants in tissue culture as an additive to the media; 2. dsRNA will be attached to positively charged clay nanoparticles and sprayed on plants in tissue culture or growth chambers; and 3. RBDV infected plants will be grafted onto transgenic plants (already developed red raspberry) that are producing RNAi silencing of RBDV. Meristems will be collected at various times after treatments, plants regenerated and tested for RBDV.
A successful berry breeding program was conducted in support of Objective 1. Plant patents were received for Eclipse, Galaxy, and Hall’s Beauty blackberry, and Echo blueberry. WSU 2166 red raspberry was released by Washington State University with ARS as a collaborator. An invention report was filed and permission to pursue a patent was granted for Twilight blackberry. Multiple selections of black raspberry, strawberry, blackberry, blueberry, and red raspberry were placed in commercial trials and resulted in several Material Transfer Research Agreements/Plant Evaluation Agreements. Substantial commercial plantings of Charm, Sweet Sunrise, and Marys Peak strawberries and Columbia Star, Columbia Sunrise, and Hall’s Beauty blackberry continue to be made, and Echo blueberries are now being sold at retail outlets. Most of the blackberry plants sold to commercial growers in the Pacific Northwest in the last five years were from this program, and over 30 percent of the total were from cultivars just released in the past five years. Over 2.5 million plants of the new strawberry releases (Sweet Sunrise, Charm, and Marys Peak) were planted in the past few years. The primocane fruiting raspberries, Vintage and Kokanee, are being grown commercially in the U.S. Kokanee, in particular, showed promise in trials in Central America, Mexico, and Europe. Over 500,000 plants of the floricane fruiting red raspberry cultivars co-developed with Washington State University were planted in the Pacific Northwest in the past five years. Characterization of black raspberry (Rubus occidentalis) germplasm that began with the collection of germplasm in the midwestern and southern U.S. has identified sources of resistance to aphids and verticillium wilt; molecular markers were used to identify resistance in progenies as opposed to time-consuming aphid screening. Selections from our efforts to incorporate novel Rubus species are approaching commercialization with a selection that can trace to new accessions from R. georgicus performing very well and potentially moving to grower trials in 2020 (Sub-objective 2A). Molecular markers were validated in black raspberry populations for two sources of aphid resistance (Sub-objective 2B). These populations with aphid resistance are now in the field for evaluation to identify selections with commercially important traits. Selections that are several generations removed from “wild” species, particularly R. coreanus, R. parvifolius, R. georgicus, and R. caucasicus, are close to being commercially viable (Sub-objective 2A). Work on a large, multi-state (New York, North Carolina, Washington, and Ohio) project to evaluate phenotypic performance of mapping populations and the genomic structure of black raspberry has been published and this will speed up the development of black raspberry cultivars with multiple sources of aphid resistance (Sub-objective 2B). In regard to Objective 3, ARS researchers at Corvallis, Oregon, continued efforts to characterize raspberry leaf curl disease with high-throughput sequencing (HTS) of diseased plant material collected in 2017 and 2018 from northeastern and the upper midwest areas of the U.S. Three novel viruses were identified, one has been fully sequenced by HTS and sequences confirmed by traditional sequencing. The other two have HTS data with partial confirmation by traditional sequencing. The fully sequenced virus is a strain of Raspberry vein chlorosis virus, which has been considered a European virus. One of the partially characterized viruses is a member of the Luteoviridae family of plant viruses. All three viruses are aphid transmitted and have transmission properties similar to those reported for the causal agent(s) of raspberry leaf curl disease in the 1940s, when the disease was prevalent in upper midwest and northeastern U.S. In the initial survey work carried out in 2018, native and commercial raspberries and blackberries were collected in Pennsylvania, Maine, Maryland, Minnesota and Wisconsin. Total nucleic acids were extracted from the samples and stored at -80C for use in virus testing. In the early testing to date, luteovirus is the most widespread in the region under study. The other two viruses, both members of the Rhabdoviridae family, are currently being tested using the archived nucleic acid extracts. Further virus collections are being carried out this summer in New York, Michigan, plus other growing regions on the U.S. (Southeast, Mid-Atlantic, Northwest, and California). Thus far, one of these viruses has been found in a single sample from native raspberry in Wisconsin and is considered a low priority for further work, other than continued testing in the survey. The diagnostics for the other two novel viruses have been added to the virus testing program that is being carried out in the National Clean Plant Network to ensure they are not present in material that enters raspberry and blackberry certification programs. ARS researchers at Corvallis, Oregon, have sequenced the genomes of seven raspberry cultivars infected with Rubus yellow net virus (RYNV), with the goal of examining incorporation sites into the host genome. The initial analysis suggests that the virus has one or few incorporation sites per cultivar. Also, the sequence analysis thus far has not identified any insertions of complete viral genomes. Direct evidence of the insertion of virus sequences into the host genome complicates virus testing that is carried out for certification or quarantine purposes. Efforts are underway to determine if a test can be developed that will detect RYNV when it is present as an infectious agent, but not when present as inserted sequences in the host genome. Researchers at Corvallis, Oregon, continue efforts toward the eradication of Blueberry fruit drop associated virus from blueberry fields in the U.S. In a survey for the virus in blueberry production areas of the U.S., more than 2,500 samples were tested and the virus was only detected in northern Washington. Growers have removed infected plants and treated stumps with herbicide to ensure there was no regrowth. The fields are being tested annually with infected plants removed to eliminate the virus. We are not sure how long the latent period is for the virus, so continued testing and removal for several years is required to ensure the virus has been eradicated. The virus was not detected in native vegetation near fields or ground cover within fields, suggesting that removal of infected bushes should eliminate the virus. Researchers at Corvallis, Oregon, continue work on improving virus elimination efficiency for developing ‘clean plants’ as part of the National Clean Plant Network. The test viruses for this project are Raspberry bushy dwarf virus and Strawberry necrotic shock virus, which are the most difficult viruses to eliminate using standard thermal therapy treatments. Research on the use of small interfering RNAs (RNAi) to reduce the quantity of virus in a given volume (virus titer) in plants is being tested as a foliar application and as an additive to tissue culture media. In the initial studies with RNAi, the virus titer was reduced by as much as 90%. In parallel experiments, RNAi is being combined with thermal therapy or chemotherapy, and chemotherapy is being combined with thermal therapy. Meristems have been taken from the RNAi treated plants and are being grown out to determine if reduced virus titer results in more efficient.
1. Improved diagnostics for viruses in berry crops in support of the National Clean Plant Network for Berries. The National Clean Plant Network for Berries (NCPN-Berries) has the mission to provide technologies and information on developing plants free of targeted pathogens (primarily viruses) and to facilitate the development of ‘Clean Plants’. NCPN-Berries is led by an ARS scientist in Corvallis, Oregon, and a researcher at the University of Arkansas. This team has used high-throughput sequencing (HTS) to identify six novel viruses in berry crops. Polymerase chain reaction (PCR) assays were developed for these viruses and are used for detection in certification and quarantine programs and for epidemiological studies. This collaborative work has also shown that HTS is just as effective at diagnosing viruses in berry crops as the combination of bioassays, serological assays, and PCR assays are.
2. ARS releases Twilight and Hall's Beauty blackberry. The thornless semi-erect Twilight and trailing Hall’s Beauty have expanded options for fresh and processed blackberry growers. Twilight thornless blackberry was released by ARS researchers in Corvallis, Oregon, and provides growers across the U.S. with a better option than the current standard, Triple Crown. It is in the same season and has excellent fruit quality, especially much better firmness. Hall’s Beauty thornless trailing blackberry is best suited to the Pacific Northwest and provides another option for growers. It can be grown for either the machine-harvested processed fruit market or the hand-harvested fresh fruit market.
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