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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. The mountains of Central Asia have forest species that include Malus sieversii (Ledeb.) M. Roem., a highly diverse apple crop wild relative. The Russian scientist Vavilov ex- plored the forests of Central Asia in the 1920s and made note of the wide range of M. sieversii phenotypes, suggesting that this region, includ- ing Kazakhstan, Kyrgyzstan, Tajikistan, and western China, is a center of origin for the domesticated apple (Luby et al., 2001; Wan et al., 2011; Yan et al., 2008). Malus sieversii grows in a wide range of habitats, including areas with hot summers and short winters as well as those with long, severe winters in the Tien Shan Mountains (Dzhangaliev, 2003). Wild populations are found in montane, scrub, humid as well as dry continental forests and in diverse stream habitats in xeric areas (Forsline et al., 2003). Malus sieversii trees are phenotypically diverse, offering variation in the time of flowering, quantity and quality of fruit, ripening period, fruit biochemical composition, tree architecture, winter and

The USDA-ARS National Plant Germplasm System Malus collection is maintained by the Plant Genetic Resources Unit (PGRU) in Geneva, NY. In the 1990s, a core subset of 258 trees was hand-selected to be representative of the grafted Malus collection. We used a combination of genotypic and phenotypic data to compare the diversity of the 198 diploid trees in the original core subset with that of 2114 diploid trees in the grafted field collection for which data were available. The 198 trees capture 192 of the 232 total microsatellite alleles and have 78 of the 95 phenotypic characters. An addition of 67 specific individuals increases the coverage to 100% of the allelic and phenotypic character states. Several de novo core sets that capture all the allelic and phenotypic character states in 100 individuals are also provided. Use of these proposed sets of individuals will help ensure that a broad range of Malus diversity is included in evaluations that use the core subset of grafted trees in the PGRU collection.

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.

Clonal woody crop germplasm collections often originate and are grown in distinct geographical locations. Because the degree of cold-hardiness is known to be a factor in the successful use of dormant bud cryopreservation for Malus, it was suggested that material from relatively warmer climates would not respond to cryopreservation as well as material from colder environments. To test this hypothesis, the effect of growing provenance on cryosurvival of dormant buds from three Malus (apple) cultivars grown in three locations (Geneva, NY; Davis, CA; and Corvallis, OR) was tested in 3 consecutive years. Dormant winter buds were harvested at the three locations, cryopreserved, and bud viability was tested by grafting. The collective 3-year mean viability for cryopreserved dormant apple buds for the three locations ranged from 63% to 81% of the buds surviving with the highest survival from the Corvallis site; however, the Geneva twigs were exposed to the lowest preharvest temperature. These results suggest that the temperature at the growing location may not hinder application of the dormant bud cryopreservation method with Malus to the extent previously speculated.

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’.

Seeds from wild Malus orientalis trees were collected during explorations to Armenia (2001, 2002), Georgia (2004), Turkey (1999), and Russia (1998). Seedling orchards with between eight and 171 individuals from each collection location were established at the U.S. Department of Agriculture–Agricultural Research Service Plant Genetic Resources Unit (PGRU) in Geneva, NY. Genotypic (seven microsatellite markers) and disease resistance data were collected for the 776 M. orientalis trees. The genetic diversity of the 280 individuals from Armenia and Georgia was compared with data previously published for the M. orientalis individuals from Russia and Turkey. A total of 106 alleles were identified in the trees from Georgia and Armenia and the average gene diversity ranged from 0.47 to 0.85 per locus. The genetic differentiation among sampling locations was greater than that found between the two countries. Six individuals from Armenia exhibited resistance to fire blight (Erwinia amylovora), apple scab (Venturia inaequalis), and cedar apple rust (Gymnosporangium juniperi-virginianae). The allelic richness across all loci in the individuals from Armenia and Georgia was statistically the same as that across all loci in the individuals from Russia and Turkey. A core set of 27 trees was selected to capture 93% of the alleles represented by the entire PGRU collection of 776 M. orientalis trees. This core set representing all four countries was selected based on genotypic data using a modified maximization algorithm. The trees selected for the M. orientalis core collection will be added to the main field collection at the PGRU.

ADDITIONAL INDEX WORDS. simple sequence repeat, genetic diversity, Kazakhstan, genotype, phenotype ABSTRACT. We estimate the minimum core size necessary to maximally represent a portion of the U.S. Department of Agriculture's National Plant Germplasm System apple (Malus) collection. We have identified a subset of Malus sieversii individuals that complements the previously published core subsets for two collection sites within Kazakhstan. We compared the size and composition of this complementary subset with a core set composed without restrictions. Because the genetic structure of this species has been previously determined, we were able to identify the origin of individuals within this core set with respect to their geographic location and genetic lineage. In addition, this core set is structured in a way that samples all of the major genetic lineages identified in this collection. The resulting panel of genotypes captures a broad range of phenotypic and molecular variation throughout Kazakhstan. These samples will provide a manageable entry point into the larger collection and will be critical in developing a long-term strategy for ex situ wild Malus conservation.

Genetic diversity and disease resistance are described for 496 seedlings from wild populations of Malus orientalis Uglitzh. collected in southern Russia and Turkey in 1998 and 1999. Eighty-five half-sib families were genotyped using seven microsatellite markers, and disease resistance was determined for apple scab (Venturia inaequalis Cooke), cedar apple rust (Gymnosporangium juniperi-virginianae Schwein), and fire blight (Erwinia amylovora Burrill). Individuals from the two Russian Caucasus collection locations were homogeneous compared with populations from the four Turkish collection locations. Within three of the Turkish collection locations, some half-sib families were highly diverse and several of these families had unusually high levels of disease resistance. In all, twenty individuals exhibited resistance to all three diseases. Bayesian analyses of the population structure revealed six distinct clusters. Most of the individuals segregated into two clusters, one containing individuals primarily from southern Russia and the other containing individuals from both Russia and northern Turkey. Individuals in the four small clusters were specific to Turkish collection locations. These data suggest wild populations of M. orientalis from regions around the Black Sea are genetically distinguishable and show high levels of diversity.

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.

The year 2005 marked the 25th anniversary of the establishment of the U.S. Department of Agriculture (USDA) National Plant Germplasm System (NPGS), repositories devoted to clonally propagated, horticultural fruit and nut crops. During this quarter century, facilities in Hilo, Hawaii; Mayaguez, PR.; Miami, Fla.; and Riverside, Calif. were developed to preserve collections of tropical and subtropical fruit and nut crops; facilities in Brownwood, Texas; Corvallis, Ore.; Davis, Calif. and Geneva, N.Y. preserve the temperate crops. Each of these facilities now has internationally recognized, globally diverse collections of genetic resources for their assigned genera. Germplasm of unique genotypes are maintained as growing plants, evaluated for phenotypic and genotypic traits, documented in a national public germplasm database, and freely distributed as clonal propaggules to researchers and other germplasm users around the world. Seed collections represent wild populations for some crop relatives. These 8 genebanks maintain 30,000 accessions representing 1600 species of fruit and nut crops and their wild relatives. The genebanks distribute more than 15,000 accessions annually to international researchers. Although originally conceived as working collections for crop improvement, NPGS genebanks have also become invaluable in providing the raw materials for basic plant genetic research, reservoirs for rare or endangered species or vulnerable landraces, archives of historic cultivars, and field classrooms for educating the public. These collections preserve botanical treasures as well as the American horticultural heritage for now and for future generations.

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.

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.

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.

The National Plant Germplasm System (NPGS) of the U.S. Department of Agriculture (UDSA), Agricultural Research Service (ARS), has greatly expanded since 1980. Foremost in this expansion was the addition of seven repositories for clonally propagated fruit and specialty crops. Many collections at state agricultural experiment station sites were in jeopardy as breeders retired. These collections can now be preserved by the NPGS. The NPGS has provided funding for plant exploration and exchange. From 1980 to 2004, 37 exploration/exchange proposals for fruit crops were funded, and over 3000 accessions introduced as a result. Crop Germplasm Committees (CGCs), established for each commodity have prepared genetic vulnerability statements and prioritized collection activities. The USDA ARS, National Germplasm Resources Laboratory (NGRL), facilitates international relationships, and the USDA Animal and Plant Health Inspection Service (APHIS), National Plant Germplasm Quarantine Center (NPGQC), tests and makes pathogen-tested germplasm available. As a result of the Convention on Biological Diversity (1993) and the International Treaty on Plant Genetic Resource for Food and Agriculture (2004), the USDA now pursues germplasm collection through the establishment of bilateral agreements of mutual benefit.

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.

One of the primary progenitors of the cultivated apple is Malus sieversii L., a species native to the forested regions of central Asia. Despite the horticultural importance of M. sieversii, little is known about genetic variation in this species. In this study, allozyme diversity at 18 loci was determined for 259 seedlings belonging to 31 sib families, each consisting of the set of offspring from a different open-pollinated maternal (seed) parent. Maternal parents belonged to 14 populations from four geographic regions. Genetic diversity statistics were computed from the resulting allele and phenotype frequencies. Cluster analysis of sib families showed that there was some grouping based on geographic region, but 16 of the sib families were most closely related to sib families from other regions. Analysis of molecular variance (AMOVA) indicated that 85% of the enzyme variability was due to differences among sib families within populations and 15% was due to differences among regions. No variability could be assigned to differences among populations within regions. In addition, no alleles were found that were fixed in a region and unique to that region. These results suggest that plants belonging to M. sieversii effectively form a panmictic population. Consequently, a thorough sampling of a few large populations will efficiently capture most of the genetic diversity present in wild M. sieversii.

Phlorizin is known for its role in reducing glucotoxicity and has a long history of use in diabetes research. In addition, its contribution to the pool of total phenolics adds to the overall health benefits attributed to fruit. Phlorizin is limited to Rosaceae family plants, of which apple comprises its current commercial source; however, limited information exists regarding its biodiversity among apple taxa. A subset of 22 taxa from a core collection of apple accessions representative of the global genetic diversity of apple was used to investigate the biodiversity of phlorizin present in apple shoots and in fruit relative to total phenolic content and free radical scavenging capacity. Fruit and shoots were harvested from the USDA Plant Genetic Resources Unit in Geneva, N.Y. Validation and quantification of phlorizin was conducted using a rigorous high-pressure liquid chromatography (HPLC) procedure. Total phenolics in fruit, assayed using a Folin-Ciocalteu method and expressed as gallic acid equivalents, ranged from 227 to 7181 mg·L-1 and were strongly related to 2,2' azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) antioxidant capacity for the core collection (r= 0.778). On a molar basis, phlorizin had lower antioxidant capacity than other major phenolic compounds present in apple fruit, but was more effective than ascorbic acid. Phlorizin yield in dormant apple shoots, expressed as percent weight, ranged from 0.9% to 5.5%. A rapid, 96 well micro-plate spectrophotometric assay was also developed to aid in the screening of multiple samples for selection of high phlorizin yielding apple taxa. Spectrophotometry overestimated phlorizin content as expected, but the calibration curve between HPLC and spectrophotometry was acceptable, r2 = 0.88.

In 1998, the USDA-ARS and Cornell Univ. instituted a cooperative agreement that mobilized the resources for a jointly managed apple rootstock breeding and evaluation program. The program is a successor to the Cornell rootstock breeding program, formerly managed by Emeritus Professor of Horticultural Sciences James N. Cummins. The agreement broadens the scope of the program from a focus on regional concerns to address the constraints of all the U.S. apple production areas. In the future, the breeding program will continue to develop precocious and productive disease-resistant rootstock varieties with a range of vigor from fully dwarfing to near standard size, but there will be a renewed emphasis on nursery propagability, lodging resistance, tolerance to extreme temperatures, resistance to the soil pathogens of the sub-temperate regions of the U.S., and tolerance to apple replant disorder. The program draws on the expertise available at the Geneva campus through cooperation with plant pathologists, horticulturists, geneticists, biotechnologists, and the curator of the national apple germplasm repository. More than 1000 genotypes of apple rootstocks are currently under evaluation, and four fire blight- (Erwinia amylovora) resistant cultivars have been recently released from the program. As a service to U.S. apple producers, rootstock cultivars from other breeding programs will also be evaluated for productivity, size control, and tolerance to a range of biotic and abiotic stress events. The project will serve as an information source on all commercially available apple rootstock genotypes for nurseries and growers.