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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

Simple sequence repeats (SSRs) are highly polymorphic regions of DNA that can be used for the molecular characterization of apple (Malus) germplasm. SSR markers are sufficiently variable to distinguish between individual plants in wild Malus species. In this study, accessions of Malus hupehensis were screened for fragment length variation in PCR amplified simple sequence repeat regions of DNA. The fragment length phenotype produced by five SSR primer pairs showed no variation between two lineages of M. hupehensis collected in the Changjiang (Yangtse) River valley. One lineage was collected by E.H. Wilson in 1908 near the city of Ichang, Hubei Province. The second lineage was collected by cooperators at China's Southwest Agricultural University (SWAU) in 1997 near the city of Chongqing (Chungking). M. hupehensis Plant Introduction No. 588760 from the National Plant Germplasm System lacks provenance, but displays a fragment length phenotype identical to both the Wilson and SWAU lineages. The spread of a clone may be aided by asexual reproduction through seed, which is not uncommon in polyploid apples. Two seedlings each of 15 maternal trees from the SWAU lineage were assayed for ploidy level by flow cytometry. The DNA content per nucleus for all SWAU progeny fell within the range for triploids, 2.19 to 2.68 pg DNA/nucleus. It appears that plant explorers in China separated by almost 90 years have succeeded in sampling a single clonal lineage of M. hupehensis.

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P. Boccacci, A. Akkak, D. Torello Marinoni, G. Bounous, and R. Botta

The authors thank H. Steinkellner (Zentrum fuer Angewandte Genetik, Universitaet fuer Bodenkultur, Vienna, Austria) for providing unpublished primer sequences of SSR loci, and S.A. Mehlenbacher (Oregon State University, Corvallis, Ore.) for

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A. Belaj, G. Cipriani, R. Testolin, L. Rallo, and I. Trujillo

Nine simple-sequence-repeat (SSR) primer pairs were assayed in 35 Spanish and Italian olive cultivars of commercial interest. All microsatellites were polymorphic, showing 5 to 13 alleles per locus (7.5 alleles per locus on average). The frequency of each alleles was generally low, with most of the alleles present at one or two cultivars. Heterozigosity ranged from 0.15 to 0.95; the discrimination power (PD) ranged from 0.30 to 0.93 (mean 0.79). The set of microsatellites analyzed discriminated all cultivars investigated. The combination of only three SSR primer pairs—UDO99-009+UDO99-043+UDO99-14—made possible the identification of all cultivars included in the study. Cluster analysis did not find differences between Spanish and Italian cultivars, but most of the cultivars from southern and central Spain grouped together. Hence, microsatellites markers are recommended for olive fingerprinting to generate a database for olive cultivar identification.

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Warren Lamboy, Christopher Alpha, Amy Szewc-McFadden, and Sharon Bleik

The cold-hardy Vitis (grape) collection at the USDA/ARS Plant Genetic Resources Unit in Geneva, N.Y., comprises ≈1300 accessions. While much of the collection has been evaluated for morphological and viticultural traits, little of it has been well-characterized genetically. Lack of genetic information hampers the identification of accessions, the determination of genetic relationships among them, the evaluation of potential new accessions, and the construction of a core subset of the collection. Because simple sequence repeat DNA polymorphisms (SSRs or microsatellites) have already been proven to be useful genetic markers in Vitis vinifera (non-cold-hardy wine, raisin, and table grapes), our research focuses on the use of the markers both for the identification (“fingerprinting”) of species, hybrids, subspecies, cultivars (varieties), and accessions of cold-hardy Vitis, and for the determination of genetic relationships between these taxa. Our latest research results in this area will be presented.

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Jernej Jakse, William Martin, John McCallum, and Michael J. Havey

The commercial production of onion (Allium cepa L.) inbreds, hybrids, and open-pollinated (OP) cultivars would benefit from a robust set of molecular markers that confidently distinguish among elite germplasms. Large-scale DNA sequencing has revealed that single nucleotide polymorphisms (SNPs), short insertion-deletion (indel) events, and simple sequence repeats (SSRs) are relatively abundant classes of codominant DNA markers. We identified 398 SNPs, indels, and SSRs among 35 elite onion ulations and observed that all populations could be distinguished. Phylogenetic analyses of simple-matching and Jaccard's coefficients for SSRs produced essentially identical trees and relationships were consistent with known pedigrees and previous marker evaluations. The SSRs revealed that elite germplasms from specific companies or breeding programs were often closely related. In contrast, phylogenetic analyses of SNPs and indels did not reveal clear relationships among elite onion populations and there was no agreement among trees generated using SNPs and indels vs. SSRs. This discrepancy was likely due to SNPs and indels occurring among amplicons from duplicated regions (paralogs) of the onion genome. Nevertheless, these PCR-based markers will be useful in the quality control of inbred, hybrid, and OP onion seed lots.

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Jeremiah D. Lowe and Kirk W. Pomper

Pawpaw [Asiminatriloba (L.) Dunal] is a tree fruit native to areas in the Midwest and Southeast United States. Since 1994, Kentucky State University (KSU) has served as the USDA National Clonal Germplasm Repository, or gene bank, for pawpaw; therefore, the assessment of genetic diversity in pawpaw is an important research priority for the KSU program. There are over 1800 pawpaw accessions (trees) from 16 different states and over 40 cultivars that are planted on 8 acres at the KSU farm. The objectives of this study were to develop microsatellite markers for pawpaw, and to then use those markers to evaluate 19 cultivars in the repository collection. Leaves of the pawpaw cultivar Sunflower were sent to Genetic Information Systems (Chatsworth, Calif.) for simple sequence repeat (SSR) primer and marker development. A total of 34 microsatellite primers were developed for pawpaw. These primers were then used in a preliminary screening with five pawpaw cultivars (`Sunflower', `Mitchell', `Sweet Alice', `Overleese', and `Prolific'). Results from this preliminary screening indicate that four of the primers failed to amplify any product, 12 primers were monomorphic, and 18 primers were polymorphic. Eleven additional cultivars were then screened, which produced numerous polymorphic products. For example, Primers B3 and B118 produced products ranging in size from 490 to 350 bp. Polymorphic products will be used to examine genetic variation among the pawpaw cultivars screened.

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Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

To comprehend genetic identity and relatedness in Malus germplasm held in situ and ex situ, we are employing simple sequence repeat (SSR) DNA fragment information in combination with passport and horticultural data. SSRs offer certain advantages for characterizing large arrays of germplasm efficiently. They are abundantly dispersed throughout plant genomes and are exceedingly polymorphic. In addition, they can be PCR-amplified and detected by automated fluorescence-based technology. A size-fractionated DNA library of M. ×domestica cv Golden Delicious was screened to identify SSR loci. Eight loci were found to be reliably informative and were used to prepare locus-specific primer pairs. Characterization of the 75 M. ×domestica accessions included in the core subset of the USDA-ARS Malus germplasm collection revealed six of the eight loci were polymorphic within the array. The number of alleles per locus ranged from two to 21. Throughput was enhanced by multiplexing, allowing simultaneous use of two or three primer pairs. With improved genetic characterization of Malus germplasm, we intend to better develop and relate the core subset to the rest of the collection and to in situ Malus genetic resources. SSR markers appear to be an efficient and reliable tool to expedite this process.

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P. Martínez-Gómez, S. Arulsekar, D. Potter, and T.M. Gradziel

The genetic relationships among peach [Prunus persica (L.) Batsch], almond [P. dulcis (Mill.) D.A. Webb or P. amygdalus (L.) Batsch] and 10 related Prunus species within the subgenus Amygdalus were investigated using simple sequence repeat (SSR) markers. P. glandulosa Pall. was included as an outgroup. Polymorphic alleles were scored as present or absent for each accession. The number of alleles revealed by the SSR analysis in peach and almond cultivars ranged from one to three whereas related Prunus species showed a range of one to 10 alleles. Results demonstrated an extensive genetic variability within this readily intercrossed germplasm as well as the value of SSR markers developed in one species of Prunus for the characterization of related species. Mean character difference distances were calculated for all pairwise comparisons and were used to construct an unrooted dendogram depicting the phenetic relationships among species. Four main groups were distinguished. Peach cultivars clustered with accessions of P. davidiana (Carr.) Franch. and P. mira Koehne. The second group contained almond cultivars. A third group included accessions of P. argentea (Lam) Rehd., P. bucharica Korschinsky, P. kuramica Korschinsky, P. pedunculata Pall, P. petunikowii Lits., P. tangutica (Spach) Batal., and P. webbii (Spach) Vieh.. P. glandulosa and P. scoparia Batal. were included in a fourth group.

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Eric T. Stafne, John R. Clark, Courtney A. Weber, Julie Graham, and Kim S. Lewers

Interest in molecular markers and genetic maps is growing among researchers developing new cultivars of Rubus L. (raspberry and blackberry). Several traits of interest fail to express in seedlings or reliably in some environments and are candidates for marker-assisted selection. A growing number of simple sequence repeat (SSR) molecular markers derived from Rubus and Fragaria L. (strawberry) are available for use with Rubus mapping populations. The objectives of this study were to test 142 of these SSR markers to screen raspberry and blackberry parental genotypes for potential use in existing mapping populations that segregate for traits of interest, determine the extent of inter-species and inter-genera transferability with amplification, and determine the level of polymorphism among the parents. Up to 32 of the SSR primer pairs tested may be useful for genetic mapping in both the blackberry population and at least one of the raspberry populations. The maximum number of SSR primer pairs found useable for mapping was 60 for the raspberry population and 45 for the blackberry population. Acquisition of many more nucleotide sequences from red raspberry, black raspberry, and blackberry are required to develop useful molecular markers and genetic maps for these species. Rubus, family Rosaceae, is a highly diverse genus that contains hundreds of heterozygous species. The family is one of the most agronomically important plant families in temperate regions of the world, although they also occur in tropical and arctic regions as well. The most important commercial subgenus of Rubus is Idaeobatus Focke, the raspberries, which are primarily diploids. This subgenus contains the european red raspberry R. idaeus ssp. idaeus L., as well as the american black raspberry R. occidentalis L. and the american red raspberry R. idaeus ssp. strigosus Michx. Interspecific hybridization of these, and other raspberry species, has led to greater genetic diversity and allowed for the introgression of superior traits such as large fruit size, fruit firmness and quality, disease resistance, and winter hardiness.

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Stan C. Hokanson, Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

A diverse collection of 133 Malus species and hybrids from the USDA Plant Genetic Resources Unit's core subset collection was screened with five simple sequence repeat (SSR) primer pairs in order to determine genetic identities and overall levels of genetic variation. The number of amplification products (alleles) per locus (primer pair) in this collection ranged from 6 to 39, with some genotypes showing complex banding patterns of up to four products per locus, suggesting that duplication events may have occurred within the genome. Five primer sets unequivocally differentiated all but 10 pairs of genotypes in the collection, with seven of these 10 being pairs of the same species. Within three of the species holdings surveyed, M. honanensis, M. sargentii, and M. sikkimensis, no genetic variation was revealed with the SSR markers. The discrimination power for the combined loci in this collection was nearly one, which indicates that the likelihood of two genetically different accessions sharing the same alleles at all the loci included in this study would be nearly impossible. Coupled with results from a previous survey of M. × domestica accessions, this finding suggests that with five SSR primer pairs, the majority of the Malus holdings could be assigned a unique fingerprint identity. The average direct count heterozygosity over all loci was 0.620, ranging in value from 0.293 to 0.871 over individual loci. These heterozygosity counts will be compared with a survey of naturally occurring M. sieversii to determine whether current repository holdings are representative of the overall levels of diversity occurring in Malus. Information generated with this study, coupled with passport and horticultural data will inform curatorial decisions regarding deaccessioning of duplicate holdings and plans for future germplasm collections.