The European pine marten (Martes martes) is a medium-sized carnivore of the family Mustelidae. It is found in Europe, including the major Mediterranean islands, with the exception of most of the Iberian Peninsula and Greece, and parts of Belgium and the Netherlands, with limited distribution in the UK (Proulx et al. 2004). Habitat fragmentation and human disturbance have caused a significant decline in European populations, which are now protected under the EU habitat directive. In Italy, pine martens live mainly in high canopy coniferous and deciduous forests. They are solitary animals, meeting only to breed in late spring or early summer, with relatively low population densities. Pine martens are also difficult to observe and few studies have been conducted on the status, distribution and dynamics of Italian populations (e.g. Manzo et al. unpublished). We developed a set of 13 polymorphic microsatellite markers to elucidate patterns of colonization and dispersal of pine martens across woodlands and to gain a greater understanding of their distribution in Tuscany through non-invasive genetic analysis based on samples collected using hair traps.

A genomic library enriched for dinucleotide repeats was constructed using a pooled sample of genomic DNA extracted from muscle tissue of two road-killed pine martens with a standard phenol–chloroform procedure. Approximately 5 μg of DNA was digested with RsaI and ligated to a double-stranded adaptor made of Oligo A (5′-GGC CAG AGA CCC CAA GCT TCG-3′) and Oligo B (5′ PO4- GAT CCG AAG CTT GGG GTC TCT GGC C -3′) oligonucleotides (Edwards et al. 1996). DNA fragments between 500 and 1,500 bp were eluted from a 1.5% agarose gel and hybridized to a mixture of (CA)14 and (GA)14 biotinylated probes at a final concentration of 4 μM each and 1 mg of streptavidin-coated magnetic beads (Roche). Microsatellite enriched DNA was recovered in a magnetic stand (Dynabeads), PCR amplified using the Expand Long Template Enzyme mix (Roche) and cloned using the StabyCloning kit (Delphi Genetics) as described in Ciofi et al. (2009). A total of 288 clones were harvested, boiled for 10 min in DNase/Rnase-free distilled water and cycle-sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing products were resolved by capillary electrophoresis in an Applied Biosystems 3100 genetic analyzer and screened for the presence of microsatellites using OLIGOFACTORY (Schretter and Milinkovitch 2006). Sequences were edited using CODONCODE ALIGNER (CodonCode Corporation). One hundred and thirty recombinant DNA inserts had microsatellite sequences, of which 65 had sufficient flanking regions to design primers for PCR amplification. Primers were designed using default parameters in PRIMER3PLUS (Untergasser et al. 2007) and screened for polymorphism on 1% agarose gel using 11 tissue samples from road-killed pine martens and three hair samples plucked from live-trapped animals. Of the 22 primer pairs which amplified consistently, 16 were selected for genotyping using a panel of 21 samples (12 tissues and 9 hairs from different animals) from the province of Siena, Italy. PCR amplification was performed using forward primers labelled with FAM, HEX, PET and VIC fluorescent dyes (Applied Biosystems) in 10 μl total volume containing 10 ng of DNA, 1× reaction buffer, 1.5 mM MgCl2, 300 μM of each dNTP, 0.5 μM of each primer and 0.5 U of Taq DNA polymerase (Invitrogen). Thermal profiles consisted of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 40 s at 94°C, annealing for 40 s at locus-specific temperature (Table 1) and extension of 60 s at 72°C, with a final extension step of 5 min at 72°C. The amplicons were resolved on an Applied Biosystems 3100 genetic analyser and allele sizes scored against a GeneScan 500 LIZ size standard (Applied Biosystems) using GENEMAPPER 4.0. Microsatellite genotypes from hair samples were obtained using a multiple-tube approach (Taberlet et al. 1996). After three PCR replicates, an allele was accepted when scored at least twice. Samples were scored as homozygotes after eight positive and consistent amplifications of the same allele.

Table 1 Characteristics of 13 microsatellite loci developed for the European pine marten
Full size table

Three microsatellite loci were monomorphic. For each of the other 13 polymorphic loci, scoring error due to stuttering, allele dropout and evidence for null alleles were tested using MICRO-CHECKER version 2.2.3 (Van Oosterhout et al. 2004). Observed and expected heterozygosity, departure from Hardy–Weinberg equilibrium and genotypic linkage disequilibrium were assessed using GENEPOP version 4.0 (Rousset 2008). The mean number of alleles per locus was 6 (range 2–10) and mean observed and expected heterozygosity were 0.610 and 0.698, respectively (Table 1). No significant linkage disequilibrium and no evidence of scoring errors due to stuttering or large allele dropout were observed. Departure from Hardy–Weinberg equilibrium was detected at locus Mar06 (P < 0.01), which had evidence of null alleles. This deviation was not significant after applying a sequential Bonferroni correction to adjust critical probability values for multiple tests (Sokal and Rohlf 1994). This set of polymorphic microsatellite markers will be useful to assess patterns of population structure and dispersal of European pine martens in woodlands and fragmented habitats. Positive PCR and allele scoring of hair samples is of particular relevance for populations genetic studies and presence/absence surveys based on non-invasive sampling which have so far relied upon cross-specific marker amplification (e.g. Mullins et al. 2010).