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L.J. Grauke, T.E. Thompson, Philip Forsline, and Kim Hummer

Core subsets have been formed in several clonally propagated crops; for pear (Pyrus), strawberry (Fragaria), mint (Mentha), currant (Ribes), blackberry (Rubus), blueberry (Vaccinium), apple (Malus), and pecan [Carya illinoinensis (Wangenh.) K. Koch]. Criteria for selecting entries into each core varies, as does the use each core receives. Core subsets have been selected for each of the major collections maintained at NCGR-Corvallis (pear, strawberry, mint, currant, blackberry, and blueberry). In general, core subsets include 10% of the full collection. Entries were selected on the basis of horticultural characteristics and species representation. Management of the collection is facilitated by recognition of core entries, which are frequently distributed. The 2500 accessions of the Malus collection are represented in a core subset of 200 accessions. Of those, 100 represent the 35 known species, while 100 accessions were selected from elite clones on the basis of horticultural characteristics. The core has been successfully used to find a superior virus indicator. Entries have been propagated in test orchards in five states. The core strategy was used to compare the pecan cultivar collection to seedlings from native populations throughout the species range. The analysis revealed gaps in the ex situ collection, and may have implications for in situ conservation. A core subset (26 cultivars) was selected by stratified sampling within the geographic regions to mirror the allele frequency of the cultivar collection, consciously including extreme expressions of each horticultural trait evaluated. The availability of the diverse subset has effected management and distribution.

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Carole L. Bassett, D. Michael Glenn, Philip L. Forsline, Michael E. Wisniewski, and Robert E. Farrell Jr

Reduced availability of water for agricultural use has been forecast for much of the planet as a result of global warming and greater urban demand for water in large metropolitan areas. Strategic improvement of water use efficiency (WUE) and drought tolerance in perennial crops, like fruit trees, could reduce water use without compromising yield or quality. We studied water use in apple trees using ‘Royal Gala’, a relatively water use-efficient cultivar, as a standard. To examine whether genes useful for improving WUE are represented in a wild relative genetically close to M. ×domestica, we surveyed Malus sieversii for traits associated with WUE and drought resistance using material collected from xeric sites in Kazakhstan. This collection has been maintained in Geneva, NY, and surveyed for various phenotypes and has been genetically characterized using simple sequence repeats (SSRs). These data suggest that most of the diversity in this population is contained within a subpopulation of 34 individuals. Analysis of this subpopulation for morphological traits traditionally associated with WUE or drought resistance, e.g., leaf size and stomata size and arrangement, indicated that these traits were not substantially different. These results imply that some of the genetic diversity may be associated with changes in the biochemistry, uptake, and/or transport of water, carbon, or oxygen that have allowed these trees to survive in water-limited environments. Furthermore, genes responding to drought treatment were isolated from ‘Royal Gala’ and categorized according to the biological processes with which they are associated. A large fraction of upregulated genes from roots were identified as stress-responsive, whereas genes from leaves were for the most part associated with photosynthesis. We plan to examine expression of these genes in the M. sieversii population during water deficit in future studies to compare their patterns of expression with ‘Royal Gala’.

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Wojciech J. Janisiewicz, Robert A. Saftner, William S. Conway, and Philip L. Forsline

Blue mold of apples, incited by Penicillium expansum, causes extensive losses on stored apples worldwide. Despite the severity of this problem, apple breeders do not evaluate their crosses for resistance to this disease, because there has been little resistance to blue mold in the gene pool of the germplasm used. A new apple germplasm collection from the center of origin in Kazakhstan, maintained in Geneva, NY, and representing a much broader gene pool, was evaluated for resistance to blue mold. Apples were harvested from the Elite collection trees that were clonally propagated from budwood collected in Kazakhstan and from seedling trees originating from seeds of the same trees as the Elite budwood or from other wild seedling trees in Kazakhstan. Fruit from 83 such accessions were harvested at the preclimacteric to climacteric stage, wound-inoculated with P. expansum at 103, 104, and 105 mL−1 conidial suspension, incubated for 5 d at 24 °C, and evaluated for decay incidence and severity. Two accessions were classified as immune (no decay at 103 and 104 mL−1), four as resistant (no decay at 103 mL−1), 53 as moderately resistant (lesions less than 10 mm at 103 mL−1), and 24 as susceptible. There were positive correlations (r = 0.92, 0.86, and 0.91) between decay severity and all three inoculum levels. Our results indicate a greater genetic diversity among the Kazak apple collection than among cultivated apples as evidenced by their broad range of fruit maturity, quality, and disease resistance patterns. The immune and resistant accessions may serve as a source of resistance in breeding programs and can be useful in explaining the mechanism of resistance to blue mold in apples.

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Manfredo J. Seufferheld, Cecil Stushnoff, Philip L. Forsline, and Gerardo H. Terrazas Gonzalez

Unlike cold-hardy apple germplasm, dormant vegetative buds from cold-tender accessions require stabilization of meristematic tissue to protect against injury during desiccation and cryopreservation. Dormant buds of six apple cultivars [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. `Cox's Orange Pippin', `Einshemer', `Golden Delicious', `Jonagold', `K-14', and `Mutsu'] collected at specific intervals in 1993, 1994, and 1995 at Geneva, N.Y., were stabilized by encapsulation in 5% alginate, treated with step-wise imbibition of 0.5 to 1.0 m sucrose and 0.2 m raffinose solution, and desiccated with forced air at 0 °C. Sugar-alginate stabilization reduced injury during desiccation, increased cold-hardiness of the six cold-tender cultivars frozen to -30 °C, and improved recovery following cryopreservation of buds collected before optimal cold acclimation was attained. Sucrose tissue levels did not increase following stabilization treatment, but levels of glucose and fructose, and of an unknown disaccharide increased. This procedure used nontoxic cryoprotectants, and has potential to expand the scope of dormant bud cryopreservation to include cold-tender apple germplasm.

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Gayle M. Volk, Adam D. Henk, Christopher M. Richards, Philip L. Forsline, and C. Thomas Chao

There are several Central Asian Malus species and varieties in the USDA-ARS National Plant Germplasm System (NPGS) apple collection. Malus sieversii is the most comprehensively collected species native to Central Asia. Other taxa such as M. sieversii var. kirghisorum, M. sieversii var. turkmenorum, M. pumila, and M. pumila var. niedzwetzkyana have primarily been donated to the collection by other institutions and arboreta. We sought to determine if genetic and/or phenotypic differences among the individuals that make up the gene pools of these taxa in the NPGS exhibit unique characteristics. Genetic data, based on microsatellite analyses, suggested that the diversity within each taxa is significantly greater than that among taxa. Trait data also revealed very few differences among taxa, the primary characteristic being the dark red fruit coloration and tinted flesh color of the accessions assigned to M. pumila var. niedzwetzkyana resulting from a known single-gene mutation in anthocyanin production. We found that M. sieversii is a highly diverse species with a range in genetic and phenotypic trait variation that includes the characteristics of the other Central Asian taxa of interest. We conclude that the gene pools that comprise the accessions within the NPGS Central Asian Malus collection are highly overlapping with respect to both phenotypic traits and genotypic characters.

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Philip L. Forsline, Warren F. Lamboy, James R. McFerson, and Cecil Stushnoff

The USDA–ARS germplasm collection of cold-hardy Vitis held at the Plant Genetic Resources Unit, Geneva, N.Y., has over 1300 clonal accessions maintained as field-grown vines. Security back-up using field-grown or potted vines at remote sites or via in vitro methods is costly. Cryopreservation offers a safe, cost-effective alternative. While we routinely employ cryogenic storage of dormant buds of Malus, dormant buds of Vitis generally do not appear to tolerate the desiccation levels required by our current cryopreservation protocol. Since tolerance to desiccation and cold appear to be correlated in Vitis, we tested desiccation tolerance of 60 germplasm accessions selected from the core subset to represent a range of cold hardiness. Budwood was collected in December 1995 in Geneva, stored at –4°C in sealed bags, and systematically desiccated to 30% and 20% moisture. In some treatments, additional desiccation was imposed by slow freezing to –25°C. Microscopic examination of rehydrated buds indicated 60% of accessions tolerated desiccation as low as 20% moisture. Freeze-desiccation at –25°C after desiccation at –4°C neither increased nor decreased viability in these accessions. Only slight modification so current protocols should be necessary for cryopreservation of this class. Of the remaining accessions, 25% tolerated desiccation to 30% moisture, but 15% were intolerant to any desiccation level tested. Techniques must be developed to successfully cryopreserve both these classes of accessions.

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Briana L. Gross, Gayle M. Volk, Christopher M. Richards, Philip L. Forsline, Gennaro Fazio, and C. Thomas Chao

The U.S. Department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS), Plant Genetic Resources Unit apple (Malus) collection in Geneva, NY, conserves over 2500 trees as grafted clones. We have compared the genotypes of 1131 diploid Malus ×domestica cultivars with a total of 1910 wild and domesticated samples representing 41 taxonomic designations in the NPGS collection to identify those that are genetically identical based on nine simple sequence repeat (SSR) loci. We calculated the probability of identity for samples in the data set based on allelic diversity and, where possible, use fruit images to qualitatively confirm similarities. A total of 237 alleles were amplified and the nine SSRs were deemed adequate to assess duplication within the collection with the caveat that “sport families” likely would not be differentiated. A total of 238 M. ×domestica and 10 samples of other taxonomic groups shared a genotype with at least one other M. ×domestica individual. In several cases, genotypes for cultivars matched genotypes of known rootstocks and indicated that these accessions may not accurately represent the indicated named clones. Sets of individuals with identical genotypes and similar cultivar names were assigned to sport families. These 23 sport families, comprised of 104 individuals, may have mutational differences that were not identified using the nine SSR loci. Five of the selected markers (CH01h01, CH02d08, CH01f02, G12, GD147) overlap with sets of markers that have been used to fingerprint European apple collections, thus making it possible to compare and coordinate collection inventories on a worldwide scale.

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Gayle M. Volk, Christopher M. Richards, Ann A. Reilley, Adam D. Henk, Philip L. Forsline, and Herb S. Aldwinckle

Seeds and scionwood of Malus sieversii Lebed. have been collected from wild populations of apple trees in Kazakhstan. Seedlings and grafted trees were planted in the orchards at the U.S. Dept. of Agriculture Plant Genetic Resources Unit in Geneva, N.Y. We developed core collections to capture the genetic and phenotypic diversity represented in the trees from each of two of the Kazakhstan collection sites. These core collections capture more than 90% of the genetic diversity of the original populations, as determined using seven unlinked simple sequence repeat markers and 19 quantitative traits. Since phenotypic evaluations of these materials have been completed, the 35 trees within each population will be used as parents in crosses so that the genetic diversity in the orchard populations can be captured as seed for long-term ex situ conservation. This strategy of storing seeds, rather than maintaining costly field collections, could be applied to other collections of wild plant materials in the National Plant Germplasm System.

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Gayle M. Volk, Christopher M. Richards, Adam D. Henk, Ann Reilley, Diane D. Miller, and Philip L. Forsline

The genetic diversity of a wild Malus population collected in the Kyrgyz Republic was compared with seedlings of Malus sieversii collected in Kazakhstan. Based on microsatellite marker results, we conclude that the population of 49 individuals collected in the Kyrgyz Republic includes private alleles and this population is assigned to a common genetic lineage with M. sieversii individuals found in the Karatau Mountain range of Kazakhstan. We recommend that a subset of these individuals be included in the National Plant Germplasm System Malus collection so they may be made available to breeders, physiologists, and other scientists for further examination.

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Philip L. Forsline, Leigh E. Towill, John W. Waddell, Cecil Stushnoff, Warren F. Lamboy, and James R. McFerson

Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.