MICROSATELLITE LETTERS Development of nuclear microsatellite markers for the Lesser Blind Mole Rat Nannospalax leucodon (Rodentia: Spalacidae) Oana Paula Popa • Gabriel Bogdan CHIS ¸ AMERA • Dumitru Murariu • Luis Ovidiu Popa Received: 15 April 2014 / Accepted: 5 May 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract We describe here eight polymorphic microsat- ellite markers for Nannospalax leucodon, an exclusively subterranean and highly specialized rodent species which exhibits range contractions and population declines in Europe. The level of polymorphism was tested on 36 individuals of blind mole rat from Doubroudja region, in South-Eastern part of Romania. The number of alleles across all loci ranged from 7 to 12, with an average of 6.1 ± 0.571 alleles per locus. Observed heterozygosity ranged from 0.735 to 1. These loci will be a useful tool for the study of genetic structure and diversity of blind mole rat populations, whit immediate consequences in conser- vation strategy for these species. Keywords Spalax Á Microsatellite Á Population genetics Á Conservation genetics The Lesser Blind Mole Rat, Nannospalax leucodon (Nordmann, 1840) (subfamily Spalacinae) is a subterra- nean rodent species known as a typical inhabitant of the dry and arid grassland regions of the Eastern Mediterranean and Eastern Europe. The IUCN Red List category and criteria for the species is ‘‘data deficient’’, with populations experiencing range contractions and declines in Europe (Krys ˇtufek et al. 2012). This status is due to the crude taxonomy of the species and a taxonomic revision might reveal several cryptic species, some of which could be listed as threatened, according to IUCN. Despite this situ- ation, little molecular studies have been conducted on the species (Krys ˇtufek et al. 2012). The loci described here represent therefore a valuable molecular tool for testing hypotheses relating to phylogeography, population genetics and kinship in N. leucodon, whit immediate consequences in the conservation strategy of this species at the European and/or national scale. The microsatellite markers were isolated using the 454 FLX Titanium pyrosequencing technology according to the protocol developed by Malausa et al. (2011). Genomic DNA was isolated from 50 mg of muscle tissue from ethanol preserved specimen of N. leucodon using the Nu- cleoSpin Ò Tissue kit (Macherey–Nagel GmbH & Co. KG, Du ¨ren, Germany), according to the producer’s specifica- tions. Eight probes (TG, TC, AAC, AAG, AGG, ACG, ACAT, ACTC) were used for the production of a highly enriched DNA library which was sequenced by the 454-GsFLX Titanium chemistry. A total of 5,525 sequences containing microsatellites motifs were obtained, among which 294 pairs of primers were validated by bioinformatics tools (QDD software). We tested 150 pairs of primers in PCR reactions and retained 35 primer pairs which gave constant amplification with a PCR product of expected size. One primer in each locus was added a 5‘end M13-tail which enabled sub- sequent fluorescent labeling of the PCR product. A total of 8 loci were successfully genotyped in 36 individuals of N. leucodon from Dobroudja, Romania (44°33 0 14.15 00 N, 28°40 0 04.71 00 E). The PCR genotyping reaction was performed in a 10 lL total volume containing about 50 ng of DNA template, 10 mM Tris–HCl (pH 8.8 at 25 °C), 50 mM KCl, 0.08 % (v/v) Nonidet P40, 2.5 mM MgCl 2 , each dNTP at 0.1 mM, each primer at 0.1 lM, 0.02 lM of IRD700 or IRD800 labeled M13 primer and Oana Paula Popa and Gabriel Bogdan Chis ¸amera have contributed equally to this work. O. P. Popa Á G. B. CHIS ¸ AMERA Á D. Murariu Á L. O. Popa (&) ‘‘Grigore Antipa’’ National Museum of Natural History, Kiseleff Street, No 1, 011341 Bucharest, Romania e-mail: popaluis@antipa.ro 123 Conservation Genet Resour DOI 10.1007/s12686-014-0220-x