2009 Annual Report
1a.Objectives (from AD-416)
1. Strategically expand the genetic diversity in genebank collections and the quality and quantity of associated information for priority cold hardy grape, tart cherry, and apples and their wild relatives.
2. Conserve and regenerate priority cold hardy grape, tart cherry, and apple genetic resources efficiently and effectively, and distribute samples and associated information worldwide.
3. Strategically characterize ("genotype") and evaluate ("phenotype") priority cold hardy grape, tart cherry, and apple genetic resources for molecular markers and highly heritable horticultural and morphological traits.
1b.Approach (from AD-416)
The objectives of this project will be met by: a) Surveying existing domestic and international collections of Vitis, Malus, and Prunus (tetraploid cherry) to identify material that would refine and fill gaps in NPGS collections; b) acquiring new accessions of wild species and heirloom cultivars, and as appropriate, genetic stocks of apple, cold-hardy grape, and tart cherry that have been extensively characterized genetically; c) enlisting genetic marker and other information to refine the holdings of the preceding priority genera by de-accessioning materials that are misidentified or unnecessarily redundant; d) conserving, regenerating, and distributing cold hardy grape, tart cherry, and apple genetic resources and associated information; e) backing up primary collections of cold hardy grape, tart cherry, and apples via nursery plantings, cryopreservation, and seed samples; f) developing and applying genetic markers for phylogenetic and genetic diversity analyses, emphasizing SSRs and sequence-based markers in cold hardy grape, tart cherry, and apples; g) generating SSR fingerprints for all priority accessions (ca. 5,000) of cold hardy grape, tart cherry, and apples and use them to determine the identity, diversity, phylogeny, and systematics of these genetic resources, and to enhance the effectiveness of genetic resource management; and h) incorporate characterization data into GRIN and other databases, and evaluating in collaborative research program, highly heritable traits of cold hardy grape, tart cherry, and apples according to CGC-approved descriptors, and incorporate data into GRIN and other databases. The primary link with users will be through Crop Germplasm Committees that define crop priorities for collection and evaluation.
During the period of this report germplasm was maintained for approximately 9000 accessions of apple, grape and sour cherry. A total of 150 new accessions were added to the germplasm collections. Distributions included 170 orders for 3,583 accessions. Most of the samples were for DNA and leaf samples for DNA extraction, but multiple samples of cuttings, pollen and seeds of wild species as well were sent. Delivery of germplasm to clients and stakeholders is the primary way in which the results of this Unit’s activities are distributed to the public.
Significant progress was made in cryogenic storage of both apple and cherry. Presently 2,275 of 2,621 apple cultivars and 78 of 125 cherry cultivars are backed up in liquid nitrogen at NCGRP. In spring 2009 these were accomplished:.
1)tested viability of cherry buds stored in 2008 and re-tested viability of cherry buds stored from 1997 to 2001..
2)Developed a collaborative project with ARS virologist, Dr. Ruhui Li to test for 12 viruses from samples from the cherry field source collections and samples from the trees established from 1997 cryogenically-stored buds. We found that viruses infected the source trees in that period of time since the samples from the cryogenic sources were not infected..
3)Rescued 20 Malus accessions from cryogenic storage that had died under field maintenance;.
4)In a collaborative experiment with NPGS sites in OR and CA cryopreserved (replicated across 3 sites including Geneva) 3 apple genotypes to determine effect of locality (climatic differences) on cryopreservability.
Completed and loaded to GRIN SSR fingerprints at eight loci for the Malus domestica collection, nearly 1,300 accessions. SSR fingerprints are also being used to determine if parental alleles are being conserved in M. sieversii seedlings. Because it is both expensive and risky to maintain wild seedlings in the field indefinitely, it is important to determine if maintaining the seeds of the core collection also maintains the diversity of the trees in the core.
SSR fingerprints for grape at eight loci (loaded into GRIN last year – 15,280 data points) are being validated by fingerprinting the second vine per accession. This process is about 95% complete and will add another 15,280 data points. The fingerprints of the first and second vine will be compared. Redundant fingerprints will be investigated when the vines are in fruit. Diversity will be analyzed to discover gaps or over-representation.
Use of cryogenic storage for conservation of apple genetic resources: Maintaining apple germplasm in an orchard has many risks involved to keep the trees alive and healthy for a long time period. Climatic extremes, severe disease outbreaks and other hazards make these trees very vulnerable. Cryopreservation in collaboration with NCGRP apple is nearly complete with 2275 of the 2621 clones on inventory completed (87% of the collection backed up). In 2008 and 2009, we were able to reestablish 60 varieties that had died in the field by grafting cryopreserved buds to rootstocks. This back up system which is 50-times more cost effective than having a collection planted at a second site has long-term goals. It has also proven to be valuable in the short term when certain accessions that are very susceptible to disease occasionally die. This system allows for very rapid graft recovery of accessions that we have shown survive for 20 years in liquid nitrogen. Tests will monitor long term storage at 10-year intervals into the future.
Development of a strategy for conservation of wild apples using seed: The most cost effective way of maintaining a broad spectrum of genetic diversity of wild species is through seed storage. We collaborated with USDA-ARS National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Co on a project to determine strategies to preserve alleles (in the form of seeds) from wild seedling accessions of Malus maintained at PGRU. We produced 60,000 seeds on trees of Malus sieversii that represent two diverse sites in Kazakhstan. Seeds were obtained from controlled crosses of selected sub sets. The pollinations and harvests were made in 2004-2006. Sub sets of these seeds were germinated in fall 2007 to confirm allele recovery. In 2009, SSR fingerprints are being used to effectively determine if most of the parental alleles can be conserved in the resulting seedlings. Because it is both expensive and risky to maintain wild seedlings in the field indefinitely, it is important to determine if maintaining the seeds of the core collection also maintains the diversity of the orchard trees. This project is also important since it supplements original seed collected from the wild in Kazakhstan in 1995. Many of those sites in Kazakhstan are being depleted by rampant animal grazing.
Crosses between cultivated apple and its progenitor for mapping important traits: As new genomic tools are developed for genetic improvement of apple we have collaborated with ARS, Cornell University and New Zealand labs to develop two new genetic maps. Malus sieversii accessions that were fire blight resistant were crossed in 2002 with the susceptible cultivar ‘Royal Gala’. For two of 7 populations (GMAL 4593 and GMAL 4591) developed and maintained at PGRU, leaf samples (>200 individuals in each population) were collected and sent to an ARS lab in Kearneysville and to a New Zealand lab for genotyping. The GMAL 4593 population was grafted by PGRU staff to standard seedling rootstocks (6 reps of each) and screened under SCA 1910-21000-020-04S for fire blight resistance. We found excellent segregation for fire blight resistance within this population. With the sequencing of the Malus genome by the international community nearly complete, these genetic maps will allow for more rapid introgressing of these genes into new varieties.
Identifying genes that control fruit texture in genetically-related fruit crops, e.g., apples, pears, strawberries, cherries, etc.: A large and comprehensive set of apple genetic resources, representing the nation-wide apple industry and breeding stock, is being examined for texture characteristics (firmness, crispness, and juiciness). The individual and combined effects of the genes controlling these traits will be evaluated by gene-trait association analysis. Covering the breadth of material that will be evaluated from many sites, three powerful statistical approaches will be employed – standard QTL analysis, Pedigree Based Analysis, and association mapping. PGRU’s contribution includes approximately 70 cultivars and 90 seedlings from 3 mapping populations (30 from each) of Malus that are being harvested and sent to Wenatchee, WA in summer/fall 2009 for phenotyping. When fruit from each tree has reached the desired maturity level, 20 fruit are selected and shipped over night in padded boxes to Washington for extensive phenotyping. Evaluations of texture components will use sensorial and instrumental protocols relevant to breeding and commercial applications. All germplasm will be phenotyped at harvest, after short-term storage (2 mo), after medium-term storage (4 mo) and long-term storage (6 mo). All storage will be in regular atmosphere at 1 degree C. Fruit will also be phenotyped for texture components of firmness, crispness, and juiciness and will be measured via sensory analysis by a trained panel (WSU Food Science Dept.). Other traits measured will be harvest date, starch index, ethylene evolution, fruit weight, and fruit dimensions. The apple dataset will serve as the example for the first comparison of these approaches for their contribution to the bridging of genomics and crop improvement in perennial horticultural crops.
Identifying genes that control fruit quality in apple using advanced genetic technologies: The traits that control quality horticultural traits in apple are poorly understood. A scientist of Cornell University has initiated a project with PGRU to construct two genome framework maps: one for Malus x domestica Borkh, and the other for M. sieversii (a wild progenitor species of most cultivars of the domesticated apple). A mapping population (GMAL 4595) maintained at PGRU derived from a cross of ‘Royal Gala’ X ‘Malus sieversii – GMAL 4455’ has been used. This population was planted in spring 2004 as own-rooted seedlings. To date, DNA samples have been prepared from 188 individuals in the population and dozens of markers have been analyzed. The genetic maps constructed will be used in genomic studies focusing on traits of horticultural and/or biological importance. PGRU scientists have provided phenotypic data on fruiting intensity and flowering dates over past few years. This research work will potentially better our understanding about the apple genomes and provide tools for apple improvement.
Tool developed to simultaneously assay thousands of grape genes: Penicillum expansum and Colletotrichum acutatum cause postharvest decay of apple fruit resulting in significant economic losses during storage. However, little resistance to both pathogens exists in the domesticated apple gene pool. Therefore, a collection of wild apple (Malus sieversii) germplasm from Kazakhstan (apple center of origin), located and maintained at PGRU was evaluated by ARS scientists in Kearneysville, WV and Beltsville, MD for resistance. Fruits from over 175 Kazakh M. sieversii accessions were harvested at various stages of maturity and were wound-inoculated with conidial suspensions of P. expansum and C. acutatum. Twenty inoculated fruit per conidial concentration from each accession were incubated at 24°C for 5 (for P. expansum) or 6 days (for C. acutatum) and then evaluated for decay incidence and severity. For P. expansum, 7 accessions were classified as immune (no decay at both conidia concentrations), 38 as resistant (no decay at 103 conidia mL-1), 142 as moderately resistant (lesions <10mm at conidia 103 mL-1), and 3 as susceptible. For C. acutatum, 1 accession was categorized as immune, 12 were resistant, 97 were moderately resistant, and 65 were susceptible. Differences in individual host resistance against both pathogens were expected due to differences in fungal lifestyles exhibited by P. expansum and C. acutatum. Both resistant and immune Kazakh accessions can serve as a source of genetic material in breeding programs and may be used in molecular studies to identify the genetic component(s) of host resistance to these important postharvest pathogens.
Tool developed to simultaneously assay thousands of grape genes: A collaborative project with ARS geneticists is well under way which will result in the generation of a 10K Illumina SNP chip for grape. SNPs were derived from various sources, but predominantly by skim sequencing (1-2x coverage each) seventeen genotypes of grapes (twelve V. vinifera and five other Vitis species) by the Solexa method. The Solexa sequencing for this project is finished, from which we will select 5,000 genic and 5,000 non-genic SNPs, when blasted on to the annotated grape genome sequence. The Illumina chip made from these SNPs will then be used to genotype the entire USDA grape germplasm collection – both at Geneva and at Davis – except for those accessions classified as Vitis hybrid – at all 10,000 loci. This data will be loaded into GRIN also. Genotyping to a depth of 10,000 loci will provide us with unprecedented levels of genetic characterization of the collections that will open the doors to further studies into diversity analysis, core collection selection, possible speciation patterns, taxonomic problems in the collections, and for the V. vinifera collection, an initial glimpse into association genetics.
Identification of unique apple accessions that are useful for mechanical harvest: Retention of ripe fruit would be expected to confer considerable advantages for current production regimes, and would be critical for potential mechanical harvesting of apples. However, this trait has generally not been targeted in breeding efforts. The genus Malus includes many wild species that are anecdotally known to retain mature fruit, especially the small-fruited species commonly referred to as crabapples. Scientists from Michigan State University and Iowa State University searched through the PGRU Malus collection for interesting abscission-related traits, and identified six unique accessions that retained fruit that was obviously overripe - with relatively high internal ethylene concentrations, low starch content, low firmness, and high coloration. Abscission of these fruits could be blocked at several points, including development of the abscission zone, activation of abscission-promoting genes/enzymes, cell-cell separation, or retention due to the vasculature, which is not subject to cell degradation during abscission. In our current work with the selected Malus accessions, we are testing these possibilities by studying the anatomy and morphology of the abscission zone during season-long development of the flowers and fruit, by evaluating fruit drop in response to exogenous ethylene, and by using molecular techniques to compare the patterns of gene activities induced during abscission between the unique non-abscising genotypes and a reference.
Provision of unique genetic sources of apple, grape & tart cherries to fruit breeders and researchers for crop improvement: One of the functions of PGRU is to distribute germplasm to our user community. We distributed: a) scions and cuttings so that the user community can establish clones; b) seeds for establishing seedling populations; c) leaf samples for DNA analysis; and d) pollen for use in breeding. Distributions in FY 2009 included 170 orders for 3583 accessions. We require the user community to respond with data on the performance of the accessions in their locality giving us useful information to document on GRIN. This germplasm is available for use by qualified researchers and other bona vide users worldwide. These distributions have spearheaded many collaborative projects with scientists worldwide who are performing research in genetics and genomics that will provide new breakthroughs to the fruit industry.
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Al-Turki, S., Shahba, M., Forsline, P.L., Stushnoff, C. 2008. Biodiversity of Total Phenolics, Antioxidant Capacity, and Juice Quality in Apple Cider Taxa. Journal of Horticulture, Environment and Biotechnology. 49(6):409-417.
Volk, G.M., Richards, C.M., Henk, A.D., Reilley, A., Miller, D.D., Forsline, P.L. 2009. Novel diversity identified in a wild apple population from Kyrgyz Republic. HortScience 44:516-519.
Richards C.M., G. Volk, A.A. Reilley, A.D. Henk, D. Lockwood, P.A. Reeves, and P.L. Forsline. 2009. Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple. Tree Genetics and Genomics 5:339-347.
Richards, C.M., Volk, G.M., Reeves, P.A., Reilley, A., Henk, A.D., Forsline, P.L., Aldwinckle, H. 2009. Selection of stratified core sets representing wild apple (Malus sieversii). Journal of the American Society for Horticultural Science. 134:228-235.