A PCR-RFLP-based method to distinguish sable (Martes zibellina) and pine marten (Martes martes)

Abstract

Species identification is an important issue in conservation and a particular focus for wildlife forensics. Molecular biological methods retain a unique power to differentiate between difficult samples that lack other identifiable characteristics. The pine marten (Martes martes) and sable (Martes zibellina) are closely related species with very similar pelage characteristics and are often difficult to distinguish from each other. The sable, however, in contrast to the pine marten, remains an endangered and protected animal in China with both hunting and fur trade strictly prohibited for this species. Here, we present a polymerase chain reaction-based restriction fragment length polymorphism method for distinguishing the two species. We sequenced a 638-bp fragment of cytochrome b gene in 39 sables, 68 pine martens, and 10 stone martens and identified all variable nucleotides. A new primer pair was subsequently designed to amplify a 316-bp fragment containing restriction sites of enzyme BseG I and BamH I that are different among martens. When the fragment was cut using BseG I, the resulting restriction pattern was identical in the sable and pine marten, but differed from all other martens. When cut using BamH I, the fragment generated two diagnostic fragments in the sable which could distinguish them from pine martens. This method was valid for all haplotypes of sable and pine marten thus far identified and has high potentially applicability for the identification of the two species.