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Raul de la Rosa, Celia M. James, and Kenneth R. Tobutt

Eight microsatellite primers were used to distinguish 23 olive cultivars that were parents, or potential parents, in a Spanish breeding program. Four of these microsatellites were particularly informative and were used to check the paternity of 11 olive progenies thought to come from selfings or controlled crosses involving nonemasculated flowers. Seven progenies were found to be highly contaminated, i.e. many seedlings had unexpected alleles, and only four were found to be pure or almost pure. Almost all the contamination detected came from outcrossing, indicating that placing the pollination bags well before anthesis is important and that emasculation to avoid selfing is unnecessary. More than two nonparental alleles per primer were found in each contaminated progeny, showing that more than one cultivar caused contamination. However, the allele data are consistent with `Picual' (the main commercial cultivar growing in the area where the crosses were made) being the contaminant in 48% of the nontrue seedlings (excluding `Picual' self progenies). Some other cultivars planted near the female trees were also found to be sources of contamination. The results obtained show that microsatellite analysis is a convenient technique to assess routinely the crosses made in breeding programs and to check self-incompatibility in olive. The pure progenies identified will be useful for reliable inheritance studies in olive, which have rarely been reported in the literature.

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Riaz Ahmad, Louise Ferguson, and Stephen M. Southwick

A genomic DNA library enriched for dinucleotide (CT)n and (CA)n and trinucleotide (CTT)n microsatellite motifs has been developed from `Kerman' pistachio (Pistacia vera L.). The enrichment method based on magnetic or biotin capture of repetitive sequences from restricted genomic DNA revealed an abundance of simple sequence repeats (SSRs) in the pistachio genome which were used for marker development. After an enrichment protocol, about 64% of the clones contained (CT)n repeats while 59% contained (CA)n for CT and CA enriched libraries, respectively. In the (CT)n enriched library, compound sequences were 45% while for (CA)n it was 13.5%. In both dinucleotide enriched libraries, about 80% of the clones having microsatellites have a repeat length in the range of 10 to 30 units. A library enriched for trinucleotide (CTT)n contained <19% of the clones with (CTT)n repeats. Of the clones that contained microsatellites, 62% had sufficient flanking sequence for primer design. An initial set of 25 pairs of primers was designed, out of which 14 pairs amplified cleanly and produced an easily interpretable PCR product in the commercially important American, Iranian, Turkish, and Syrian pistachio cultivars. The efficient DNA extraction method developed for pistachio kernels and shells (roasted and nonroasted) yielded DNA of sufficient quality to use PCR to create DNA fingerprints. In total, 46 alleles were identified by 14 primer pairs and a dendrogram was constructed on the basis of that information. The SSR markers distinguished most of the tested cultivars from their unique DNA fingerprint. An UPGMA cluster analysis placed most of the Iranian samples in one group while the Syrian samples were the most diverse and did not constitute a single distinct group. The maximum number of cultivar specific markers were found in `Kerman'(4), the current industry standard in the United States, and the Syrian cultivar Jalab (5). The technique of using extracted DNA from pistachio kernal or shell coupled with the appropriate marker system developed here, can be used for analyses and measurement of trueness to type.

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Peter Boches, Nahla V. Bassil, and Lisa Rowland

Sixty-nine accessions representing wild and domesticated highbush blueberry (Vaccinium corymbosum L.) germplasm were genotyped using 28 simple sequence repeats (SSRs). A total of 627 alleles was detected and unique fingerprints were generated for all accessions. Suspected duplicate accessions of `Coville' and `Ivanhoe' had DNA fingerprints that were identical to `Coville' and `Ivanhoe', respectively. Genetic similarity measures placed wild and cultivated blueberries in separate groups. Northern highbush blueberries grouped among ancestral clones that were used extensively in blueberry breeding such as `Rubel' and `Stanley'. Southern highbush blueberries formed a separate group from northern highbush blueberries. The microsatellite markers used here show excellent promise for further use in germplasm identification, in genetic studies of wild Vaccinium L. populations, and for constructing linkage maps.

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Mario I. Buteler, Don R. LaBonte, and Robert L. Jarret

Microsatellites or simple sequence repeats (SSRs) were used to characterize 20 sweetpotato genotypes and to assign paternity for offspring from crosses among them. The PCR amplifications were performed with each of the sweetpotato genotypes and primers flanking a SSR loci previously characterized with the varieties Beauregard and Excel and 20 offspring from a cross among them. The PCR reaction products were separated in nondenaturing 12% acrylamide gels run at 25 V·cm–1 for 5 hours, and DNA fragments were visualized with silver staining. Gels were scanned on a flat bed scanner and analyzed using the Pro-RFLP software package. Three primer pairs were sufficient to produce an allelic profile capable of differentiating the 20 genotypes from each other. More than seven alleles/loci were found using each of the three primer pairs assayed. Occasionally primers produced allelic products clearly localized in two or three regions of the gel. These multiple loci segregated independently in a diploid fashion. This evidence suggests that there is not total homology among the three sweetpotato genomes.

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Gayle M. Volk, Christopher M. Richards, Adam D. Henk, Ann A. Reilley, Nahla V. Bassil, and Joseph D. Postman

Edible european pears (Pyrus communis L. ssp. communis) are derived from wild relatives native to the Caucasus Mountain region and eastern Europe. Microsatellite markers (13 loci) were used to determine the relationships among 145 wild and cultivated individuals of P. communis maintained in the National Plant Germplasm System (NPGS). A Bayesian clustering method grouped the individual pear genotypes into 12 clusters. Pyrus communis ssp. caucasica (Fed.) Browicz, native to the Caucasus Mountains of Russia, Crimea, and Armenia, can be genetically differentiated from P. communis ssp. pyraster L. native to eastern European countries. The domesticated pears cluster closely together and are most closely related to a group of genotypes that are intermediate to the P. communis ssp. pyraster and the P. communis ssp. caucasica groups. Based on the high number of unique alleles and heterozygosity in each of the 12 clusters, we conclude that genetic diversity of wild P. communis is not fully represented at the NPGS. Additional diversity may be present in seed accessions stored in the NPGS and more pear diversity could be captured through supplementary collection trips to eastern Europe, the Caucasus Mountains, and the surrounding countries.

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Suzanne L. Downey and Amy F. Iezzoni

Black cherry (Prunus serotina Ehrh.) is a common secondary forest species with a wide endemic distribution ranging from Nova Scotia south into Mexico, Ecuador, and Peru. Although planted in the United States for its valued lumber, black cherry is essentially a wild species with small fruit ≈6 to 10 mm in diameter. In contrast, in Mexico and Ecuador, domesticates of this species called Capulin, have much larger (2 to 2.5 cm in diameter) edible fruit. To date, no studies of the genetic diversity within North American black cherry or the ancestral origin of the Capulin types have been conducted. Simple sequence repeats (SSRs, also termed microsatellites) would be the marker of choice for such genetic diversity studies due to their hypervariability; however, generation of these sequence-based markers is expensive. Therefore, our objective was to determine if markers already identified in other Prunus L. species would be informative in black cherry. The black cherry germplasm screened consisted of selections originating from Michigan, Mexico, and Ecuador. A chloroplast DNA marker, originally generated from sour cherry (P. cerasus L.), amplified three different sized products in black cherry. Four of the eight nuclear SSR markers tested from peach [P. persica L. Batsch (Peach Group)], sour cherry, and sweet cherry (P. avium L.) also amplified and identified polymorphic markers. Together these four primer pairs resolved 54 putative alleles for the 66 black cherry accessions assayed. Success of the sweet cherry, peach, and sour cherry primers in identification of polymorphic markers in black cherry indicates it should be possible to use these markers for comprehensive molecular genetic studies in black cherry.

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Darush Struss, Riaz Ahmad, Stephen M. Southwick, and Manuela Boritzki

Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evaluate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite containing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of `Bing' fruit collected from different grocery stores in the United States to that of a standard `Bing' cultivar. Results indicated that, with one exception, all `Bing'samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study.

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Karen R. Harris-Shultz, Brian M. Schwartz, Wayne W. Hanna, and Jeff A. Brady

fragments Theor. Appl. Genet. 112 727 737 Boutin-Ganache, I. Raposo, M. Raymond, M. Deschepper, C.F. 2001 M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods Biotechniques 31

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Phillip A. Wadl, Robert N. Trigiano, Dennis J. Werner, Margaret R. Pooler, and Timothy A. Rinehart

sequences in phylogenetic analyses of angiosperms Ann. Mo. Bot. Gard. 82 247 277 Barbará, T. Palma-Silva, C. Paggi, G.M. Bered, F. Fay, M.F. Lexer, C. 2007 Cross-species transfer of nuclear microsatellite markers: Potential and limitations Mol. Ecol. 16 3759

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Pilar Soengas, Pablo Velasco, Guillermo Padilla, Amando Ordás, and Maria Elena Cartea

Brassica napus includes economically important crops such as oilseed rape, rutabaga, and leaf rape. Other vegetable forms of Brassica napus, namely nabicol and couve-nabiça, are grown in northwestern Spain and north of Portugal, respectively, and their leaves are used for human consumption and fodder. The relationship of nabicol with other Brassica napus leafy crops was studied before, but its origin remained unclear. The aims of this work were to study the genetic relationships among nabicol landraces and other B. napus crops based on microsatellites and to relate the genotypic differences with the use of the crop. The relationship among 35 Brassica napus populations representing different crops was studied based on 16 microsatellite markers. An analysis of molecular variance was performed partitioning the total variance into three components. The source of variation resulting from groups was defined considering the main use of the crop and accounted for a smaller percentage of variation than other sources of variation, proving that this division is not real. Populations clustered into seven different clusters using a similarity coefficient of 0.82. No clear association was evident between clusters and the main use of populations, suggesting genetic differences among populations could reflect differences in their origin/breeding or domestication. Spanish nabicol could have originated from a sample of couve-nabiças, and couve-nabiças could be used to improve nabicol landraces, because they have a narrow genetic basis that limits their potential for breeding.