Characterization of nine new microsatellite loci for the marbled newt, Triturus marmoratus.

Abstract

Triturus marmoratus is distributed throughout central, western and southern France as well as in a large portion of northern Iberia. Despite being categorized as of least concern by the IUCN, it is protected by the Annex IV of the EU Habitats Directive and the Annex III of the Bern Convention (Arntzen et al. 2009). As most amphibians, T. marmoratus is particularly sensitive to habitat loss and landscape fragmentation (Cushman 2006). Maintaining connectivity is therefore critical to ensure genetic diversity and sustainable populations. A good knowledge of the population genetic structure is necessary. Microsatellites are among the most commonly used genetic markers in numerous field of research (DeFaveri et al. 2013) and can be used in landscape genetics to assess population structure and quantify gene flow. Thus it can provide useful information for applied conservation ecology (Emel and Storfer 2015). Total genomic DNA from epithelial cells obtained from buccal swab and from tissue muscle of 15 marbled newts was isolated using the DNeasy Blood and Tissue kit (Qiagen, Valencia, USA) pooled in equal quantities, and 1 μg of the pooled DNA was used for the development of microsatellites libraries through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries as described in Malausa et al. (2011). Briefly, total DNAwas mechanically fragmented, ligated to standard adapters, enriched for AG, AC, AAC, AAG, AGG, ACG, ACAT and ATCT repeated motifs, and amplified by PCR. The purified products were then sequenced on a GsFLX PTP (Roche, Basel, Switzerland) following the manufacturer’s instructions. One hundred and seventythree microsatellite sequences were identified. From these, all sequences with an important number of repeats (as they have better chances to be polymorphic) and a size greater than 100 bp were selected. These resulted in the selection of