Molecular Ecology Notes (2005) doi: 10.1111/j.1471-8286.2005.01046.x © 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Isolation and characterization of six polymorphic microsatellite markers in the freshwater turtle Mauremys rivulata (Testudines: Geoemydidae) G. MANTZIOU,*† A. ANTONIOU,†§ N. POULAKAKIS,*† G. GOULIELMOS,‡ C. S. TSIGENOPOULOS,§ T. PINOU¶ and M. MYLONAS*† * Natural History Museum of Crete, University of Crete, Knossos Ave., PO Box 2208, 71409 Irakleio, Crete, Greece, † Department of Biology, University of Crete, Vassilika Vouton PO Box 2208, 71409 Irakleio, Crete, Greece, ‡ Department of Internal Medicine, Division of Rheumatology, Clinical Immunology and Allergy, School of Medicine, University of Crete, Voutes, 715 00 Irakleio, Greece, § Department of Genetics and Molecular Biotechnology, Hellenic Centre for Marine Research, Thalassocosmos Gournes Pediados, 71500 Irakleio, Crete, Greece, ¶ Department of Biological and Environmental Sciences, Western Connecticut State University, 181 White Street, Danbury, CT 06810, USA Abstract Six polymorphic microsatellite loci containing dinucleotide repeats were developed for the freshwater turtle Mauremys rivulata. The number of alleles ranged from five to 18. The observed and expected heterozygosities ranged from 0.19 to 0.79 and 0.46 to 0.90, respectively. These markers may serve as a valuable tool for population genetics analyses and provide information on the evolutionary history of the species. Keywords: Geoemydidae, Mauremys rivulata, microsatellites Received 22 February 2005; revision accepted 6 April 2005 The freshwater turtle Mauremys rivulata is one of the three Mediterranean species of the genus Mauremys . This species is distributed in Croatia, Serbia and Montenegro, Albania, Bulgaria, Greece, Cyprus, the Mediterranean coast of Turkey, Syria, Lebanon, Jordan and Israel (Wischuf & Busack 2001). Populations of M. rivulata have experienced a severe dec- line during the last decades due to anthropogenic pressure. Based on cytochrome b sequence data (Mantziou et al . 2004), intraspecific relationships could not be clearly resolved, since the variation within M. rivulata was relatively small and not related to the geographical origin of the specimens. Microsatellite markers may serve as a valuable tool for the investigation of the existing genetic variability and population structure. For the isolation of the microsatellite loci, already pub- lished protocols were followed, with some modifications. Total genomic DNA was extracted from nail samples using the Holmes–Bonner lysis solution (Holmes & Bonner 1973) and standard phenol–chloroform extraction. DNA was digested with the restriction enzymes Alu I, Hae III and Rsa I, and the resulting fragments, ranging from 200 to 800 bp, were ligated to double-stranded SNX linkers using high concentration DNA ligase to achieve efficient blunt-end ligation (Hamilton et al . 1999). Ligations were used without further purification for subtractive hybridization with 5 ′ - biotinylated oligonucleotides primers [(GA) 10 and (AC) 8 ] as the hybridization target, and with streptavidin-coated magnetic beads (Promega) as the substrate to capture genomic DNA-oligonucleotide hybrids (Kijas et al . 1994). Repeat-enriched DNA was rendered double-stranded and amplified in a polymerase chain reaction (PCR) using the forward SNX linker as a primer. Amplification was verified on a 2% agarose electrophoresis gel loaded with a 5- μ L aliquot. The resulting purified PCR product was then directly ligated overnight at 4 ° C into the plasmid pGEM-T vector (Promega), followed by transformation in Escherichia coli JM109 competent cells (Promega). A total of 100 colonies were screened for dinucleotide repeats using a triple PCR technique: amplification with two primers of the vector (T7 and SP6) and one nonbiotinylated primer, such as (GA) 10 or (AC) 8 . Ten positive colonies that had at Correspondence: G. Mantziou, Fax: +30 2810 324 366; E-mail: mantziou@nhmc.uoc.gr