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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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M. Dolores Loureiro, M. Carmen Martínez, Jean-Michel Boursiquot, and Patrice This

`Albariño' (Vitis vinifera L.) is an important grape cultivar in Spain, morphologically diverse but subject to much misnaming. The objectives of the present work were to correct some of the more common misnamings concerning `Albariño' and to evaluate the genetic variability within this cultivar by analyzing DNA polymorphisms using randomly amplified polymorphic DNA (RAPD) markers and microsatellite techniques. Several accessions of `Albariño' (16 accessions from Misión Biológica de Galicia, one accession from El Encin, one accession from Rancho de la Merced), related cultivars (`Alvarinho', `Caíño blanco', `Cainho branco', `Loureiro'), and cultivars presumably identical to misnomers (`Savagnin blanc' and `Gewürztraminer') were analyzed using 20 RAPD markers and six microsatellite loci. Both techniques revealed polymorphism among `Albariño', `Caíño blanco', `Albariño' from Rancho de la Merced and `Loureiro'. No polymorphism was detected among the 16 `Albariño' accessions from Galicia, the `Albariño' accession from El Encin and `Alvarinho', nor among the `Albariño' accession from Rancho de la Merced, `Savagnin blanc' and `Gewürztraminer', nor between `Caíño blanco' and `Cainho branco'. These results enabled us to clarify the main misnomers concerning these cultivars. The absence of polymorphism among the true `Albariño' accessions did not allow the detection of any clonal variation. The suitability of both techniques for defining the cultivar level for grapevine is discussed.

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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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A. Vainstein and H. Ben-Meir

Mini- and microsatellite probes were hybridized to DNA of 24 rose (Rosa×hybrida) genotypes. The resultant DNA fingerprints were shown to be genotype-specific, thereby enabling cultivar identification at the DNA level. Restriction enzyme Dra I yielded the most informative band patterns. Full-sib family analysis of DNA fingerprints revealed 32 parental-specific bands out of the 128 observed in the parents. These bands were revealed cumulatively by phage (M13), human (33.6), and oligonucleotide (GACA)4 probes. Only one pair of these loci was found to be allelic, and no linked pairs were detected in the progeny analyzed. The probability of two offspring from this cross having identical DNA fingerprints was calculated to be 2 × 10-8.

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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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E.J. Parks, J.W. Moyer, and J.H. Lyerly

Fluorescent amplified fragment length polymorphism (F-AFLP) and microsatellites (SSRs) were used to evaluate new guinea impatiens (Impatiens hawkeri W. Bull) cultivars. Ninety-five quality-selected polymorphic fragments from 10 F-AFLP+3 primer combinations were used to evaluate 100 cultivars representing a variety of colors, forms, and breeding programs. Jaccard similarities and unweighted pair-group method of the arithmetic average (UPGMA) clustering formed a dendrogram with three cultivar groups, to a large extent clustering the cultivars by breeder with a high cophenetic correlation coefficient. A small insert genomic library was created and 442 kb of new guinea impatiens sequence was screened for repetitive motifs, resulting in 14 microsatellite markers. A subset of 46 cultivars representing five commercial breeding companies and 11 cultivar series was selected for microsatellite analysis. Seven loci were polymorphic, with two to six alleles per locus. Although both methods were equally effective in distinguishing the cultivars from one another, the topologies of the dendrograms for the two methods were different. The topology of the AFLP dendrogram reflected possible relationships based on cultivar series and breeding company, while the SSR dendrogram did not. The objectives of this research were to develop and validate both F-AFLP and SSR methodologies for new guinea impatiens, identify markers that can be reliably used for fingerprinting, and create a database for future cultivar comparisons.

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Xiaobai Li, Weirui Li, Chenlu Di, Ming Xie, Liang Jin, Cheng Huang, and Dianxing Wu

tetranucleotide microsatellites for fine-scale discrimination among endangered chinook salmon ( Oncorhynchus tshawutscha ) Mol. Ecol. Notes 3 376 379 Hayes, B. Sonesson, A.K. Gjerde, B. 2005 Evaluation of three strategies using DNA markers for traceability in