Molecular Ecology Notes (2006) 6, 197–200 doi: 10.1111/j.1471-8286.2005.01191.x © 2006 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria × ananassa A. MONFORT,* S. VILANOVA,* T. M. DAVIS† and P. ARÚS* * Laboratori de Genètica Molecular Vegetal CSIC-IRTA, Departament de Genètica Vegetal, Carretera de Cabrils s/n. 08348 Cabrils, Barcelona, Spain, † Department of Plant Biology, University of New Hampshire, Durham, New Hampshire 03824, USA Abstract A set of 41 polymorphic microsatellite markers were developed using a CT/AG-enriched genomic library of Fragaria vesca cv. Reine des Vallées. Thirty-five of them were polymorphic in F. vesca and were tested in one accession each of six additional diploid Fragaria species and the octoploid Fragaria × ananassa. A mean of 5.3 alleles per locus and a low level of observed heterozygosity were generally detected in the 32 single-locus simple sequence repeats of F. vesca. Most of these loci amplify in the other diploid species and in F. × ananassa. Keywords: microsatellites, transportability, variability Received 3 August 2005; revision accepted 12 September 2005 Fragaria vesca , a wild strawberry species native to the temperate regions of Eurasia and North America, is one of the 20 described species of Fragaria (Rosaceae). The ploidy level in this genus varies from diploid ( F. vesca ) to octoploid (e.g. the cultivated strawberry, Fragaria × ananassa ). Simple sequence repeats (SSRs) have already been developed in Fragaria diploid species (Sargent et al . 2003; Cipriani & Testolin 2004; Hadonou et al . 2004; Lewers et al . 2005) and used for variability analysis and map construction. In this paper, we report a new set of SSR (or microsatellite) markers developed in F. vesca that were studied for varia- bility in 14 genotypes: one of the octoploid cultivated strawberry (‘8603’); and 13 of diploid species, including eight of F. vesca (‘Reine des Vallées’, ‘Baron Solemacher’, ‘Yellow Wonder’, ‘FDP815’, ‘WC6’, ‘Pawtuckaway’, ‘GS2C’ and ‘DN1C’) two of Fragaria nubicola (‘CFRA 520’ and ‘FDP601’), and one of each of Fragaria mandshurica (‘GS99’), Fragaria viridis (‘CFRA 333’), Fragaria iinumae (‘CFRA 377’) and Fragaria nilgerrensis (‘CFRA 1358’). A microsatellite-enriched genomic library for AG/TC motifs was constructed using DNA from F. vesca ‘Reine des Vallées’ following the method based on biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles described in Aranzana et al . (2002). DNA from young leaves was extracted using the method of Doyle & Doyle (1990). After Eco RI digestion, DNA was ligated to an Eco RI adapter, and the (CT) n -enriched small fragments obtained were ligated to the pGem-T easy vector, and transformed into JM109 Competent Cells (Promega). Inserts from recombinant clones were polymerase chain reaction (PCR)-amplified using standard M13 primers, transferred onto HybondN+ nylon membrane and hybridized with the oligonucleotide (AG) 15 , labelled with γATPP 33 . The clone analysis showed that 50% of them had the microsatellite. DNA from 88 recombinant colonies con- taining the (CT) n microsatellite and randomly chosen was sequenced. A set of flanking primer pairs was designed for each of 41 sequences containing a unique SSR motif. The remaining sequences were discarded due to redundancy, short motif (less than 8 repeats) or extreme localization of the microsatellite. Primer pairs (Table 1) were designed to be 18–22 bp long with an annealing temperature between 45 and 65 ° C (optimum 60 ° C) and to give an expected product size of 100 – 200 bp, using the program primer 3 (http://www. Correspondence: Amparo Monfort, Fax: +34 93 7533954; E-mail: amparo.monfort@irta.es