The edible dormouse (Glis glis) (Rodentia: Gliridae) is largely widespread in western, central, and southeastern Europe and western Asia (Storch 1978). This hibernating arboreal rodent is especially influenced by habitat fragmentation and human activities. In Poland, where edible dormouse is endangered and protected by law (Pucek and Jurczyszyn 2001), some efforts have been made to develop management plans for Glis glis that include monitoring, protection, and reintroduction of this species. All these activities could be monitored and helped by the use of polymorphic DNA markers. Despite the efforts that were done to develop microsatellite markers for this species (Hürner et al. 2009; Dabert et al. 2009; Segelbacher et al. 2010), still there was a need of new markers due to a relatively low level of polymorphism of the published loci, which displayed no more than 7 alleles per locus. Here we present new fourteen polymorphic microsatellite markers for dormice, including four loci exhibiting from 10 to 31 alleles per locus.

Total genomic DNA for this study was extracted from a liver tissue of the edible dormouse specimen died during hibernation in the Zoological Garden in Poznan, 1997. After digestion with HaeIII and AluI (NEB, New England Biolabs, USA) DNA was treated with MBN nuclease (NEB) and FastAP alkaline phosphatase (Fermentas, Lithuania). Then, column-purified DNA fragments were ligated to the double-stranded SNX linkers as described by Hamilton et al. (1999). Ligation success was monitored by PCR with SNX forward primer. Ligation mixture was denatured at 95 °C for 5 min and hybridized to 5′-biotinylated (CA)12 and (CT)12 oligonucleotides at 60 °C for 1 min, and then incubated in rotation at 60 °C for 15 min with streptavidin-coated paramagnetic beads (Dynabeads, Life Technologies, USA). Repeat-enriched fragments were PCR amplified using SNX forward primer and cloned into pCR4-TOPO vector using TOPO TA Cloning Kit (Life Technologies). After screening, 96 positive clones were sequenced on an ABI 3130XL DNA analyzer (Applied Biosystems). After excluding clones with <15 repeats, we found 27 non-redundant microsatellite sequences flanked by regions sufficient to design primers. However, among primer sets developed for these loci, only 14 gave specific PCR products and/or were polymorphic in 15 tested specimens representing Croatian and Polish populations (Table 1).

Table 1 Characterization and amplification conditions of 14 STR markers developed for edible dormice
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The microsatellites were tested for polymorphism on 427 individuals representing one population from Croatia (48) and several populations from Poland (329). PCR amplifications with fluorescently labeled primers were carried out in 6 μl reactions containing Type-it Microsatellite Kit (Qiagen, Germany), 0.2 μM each of primers, and 10–100 ng of template DNA using program as follows: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, annealing temperature (see Table 1) for 90 s, 72 °C for 30 s, and final extension at 65 °C for 45 min. M13-tailed loci (Oetting et al. 1995) were amplified at the same conditions, except the concentration of primers, which was 0.15 μM for forward primer, 0.25 μM for fluorescently labeled M13 primer, and 0.4 μM for reverse primer. We found that four loci (23pilch, 24pilch, 30pilch, and 36pilch) could be amplified in a multiplex PCR under the conditions as described above. The amplified alleles were separated on an ABI PRISM 3130XL with Genescan 600LIZ size standard and scored in GeneMapper 4 (Applied Biosystems).

Hardy–Weinberg equilibrium (HWE) and genotypic disequilibrium between pairs of loci were tested using GENEPOP 4.0 (Rousset 2008). Number of alleles, and expected and observed heterozygosities were estimated in GENALEX 6 (Peakall and Smouse 2006). Each locus was tested for null allele frequencies using Micro-Checker (van Oosterhout et al. 2004). The number of alleles per locus in all analyzed specimens ranged from 4 to 31 (Table 2), with a mean of 10. This relatively high level of polymorphism was found in both Polish and Croatian populations (a mean of 8 and 7 alleles per locus, respectively). The remaining statistical parameters were tested on the Croatian population only, because it showed a more equilibrated structure than strongly isolated populations from Poland. We found no evidence of linkage disequilibrium between loci. In two loci, 37pilch and 47pilch, significant evidences for null alleles were found with 95 % confidence interval. Only one locus (26pilch) demonstrated significant deviations from HWE in the sampled population (P value = 0.005), and exhibited deficit in heterozygotes. This deviation from expectations could be a result of insufficient sample size of the tested Croatian population.

Table 2 Statistical parameters of 14 STR markers in dormouse populations
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These new highly polymorphic microsatellite loci in combination with previously published markers (Hürner et al. 2009; Dabert et al. 2009; Segelbacher et al. 2010) could provide a powerful tool for detailed genetic studies of this endangered species.