Molecular Ecology Resources (2008) 8, 1457–1459 doi: 10.1111/j.1755-0998.2008.02217.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Eight new tetranucleotide microsatellite loci for the agile frog (Rana dalmatina) J. S. HAUSWALDT*,† J. FUESSEL,† J. GUENTHER‡ and S. STEINFARTZ§ *Max-Planck Institute for Evolutionary Biology, 24306 Plön, Germany, †Unit of Evolutionary Genetics, Institute for Genetics, University of Cologne, 50674 Cologne, Germany, ‡Institute of Zoology, University of Veterinary Medicine, 30559 Hannover, Germany, §Group of Molecular Ecology and Behaviour, University of Bielefeld, 33615 Bielefeld, Germany Abstract We describe eight new polymorphic tetranucleotide microsatellite loci isolated from the agile frog (Rana dalmatina). In 25 individuals from the Nature Reserve Lüneburger Heide (Lower Saxony, Germany), the number of alleles per locus ranged from four to nine and average observed heterozygosities from 69.1% to 80.7%. No evidence for linkage disequilibrium was found and none of the loci showed significant deviation from Hardy–Weinberg expecta- tions. These microsatellite DNA markers are suitable tools for addressing population genetics issues in this endangered species. Keywords: anura, microsatellites, motif, Rana dalmatina, tetranucleotide Received 20 January 2008; revision accepted 9 March 2008 The agile frog (Rana dalmatina) is widespread in Europe, ranging from eastern Spain to Romania, and from Denmark to southern Greece. In Germany, the agile frog has a rather disjunct distribution: its largest occurrences are in central and southern Germany. In northern Germany, its distri- bution is very patchy: it is found only in two coastal areas on the Baltic Sea (Rügen and Darß), north of the Harz Mountains, and in the Lüneburger Heide, a heathland area north of Hanover (Günther 1996). Rana dalmatina is listed as an endangered species within the Fauna-Flora-Habitat (FFH) directive appendix IV. During the course of a larger project launched to establish standardized DNA markers as a set of species-specific microsatellite loci for threatened German amphibian species, we describe here the isolation of polymorphic tetranucleotide microsatellite loci for R. dalmatina. These new microsatellite loci will be helpful to address questions at the interface of FFH-relevant moni- toring and conservation issues. From our own experience (Steinfartz et al. 2004; Hauswaldt et al. 2007), we have found microsatellite loci with tetranucleotide motifs easier to score and therefore aimed at isolating tetranucleotide loci. For the enrichment, genomic DNA was used from an individual R. dalmatina collected near Cologne, Germany. DNA was enriched with two oligo mixtures containing tetranucleotide probes following the protocol by Glenn & Schable (2005). Mix A contained (AAGT) 8 (AGAT) 8 (AACT) 8 (ACAT) 8 and (AAAT) 8 as probes, mix B contained (AAAC) 6 (AAAG) 6 (AATC) 6 (ACAG) 6 (ACTC) 6 and (ACTG) 6 . In brief, genomic DNA was digested with RsaI, ligated to SuperSNX linkers, hybridized to biotinylated microsatellite oligonucleotides and captured with Dynabeads (Dynal Biotech Inc.). Unspe- cifically bound DNA was washed away according to the protocol and specifically bound DNA was recovered by polymerase chain reaction (PCR) with the SuperSNX-f (5′-GTTTAAGGCCTAGCTAGCAGAATC-3′) primer. PCR products were enriched for the specific microsatellite motifs (see above) an additional time using the same procedure and conditions as described before. The resulting PCR products were cloned using the TOPO TA Cloning System (2.1) (Invitrogen). Inserts from colonies were amplified using M13 primers. Altogether, 65 colony-PCR products of 500–1000 bp were sequenced with T3 and T7 primers using BigDye version 3.1 chemistry (Applied Biosystems) on an ABI 3730 automated sequencer. Of these, 57 were from the enrichment with mix A and eight from the enrichment with mix B. Forty clones contained microsatellite motifs with at least six repeats units. Fifteen of these were not considered for further analysis as they contained mixtures of di- and Correspondence: J. S. Hauswaldt, Unit of Evolutionary Biology, Institute of Zoology, Technical University of Braunschweig, Spiel- mannstrasse 8, 38106 Braunschweig, Germany. Fax: +49 (0) 531 391 8198; E-mail: s.hauswaldt@tu-bs.de