title: Development of Nuclear Microsatellite Loci and Mitochondrial Single Nucleotide Polymorphisms for the Natterjack Toad, Bufo (Epidalea) calamita (Bufonidae), Using Next Generation Sequencing and Competitive Allele Specific PCR (KASPar).
abstract: Amphibians are undergoing a major decline worldwide and the steady increase in the number of threatened species in this particular taxa highlights the need for conservation genetics studies using high-quality molecular markers. The natterjack toad, Bufo (Epidalea) calamita, is a vulnerable pioneering species confined to specialized habitats in Western Europe. To provide efficient and cost-effective genetic resources for conservation biologists, we developed and characterized 22 new nuclear microsatellite markers using next-generation sequencing. We also used sequence data acquired from Sanger sequencing to develop the first mitochondrial markers for KASPar assay genotyping. Genetic polymorphism was then analyzed for 95 toads sampled from 5 populations in France. For polymorphic microsatellite loci, number of alleles and expected heterozygosity ranged from 2 to 14 and from 0.035 to 0.720, respectively. No significant departures from panmixia were observed (mean multilocus FIS= -0.015) and population differentiation was substantial (mean multilocus FST= 0.222, P < 0.001). From a set of 18 mitochondrial SNPs located in the 16S and D-loop region, we further developed a fast and cost-effective SNP genotyping method based on competitive allele-specific PCR amplification (KASPar). The combination of allelic states for these mitochondrial DNA SNP markers yielded 10 different haplotypes, ranging from 2 to 5 within populations. Populations were highly differentiated (GST= 0.407, P < 0.001). These new genetic resources will facilitate future parentage, population genetics and phylogeographical studies and will be useful for both evolutionary and conservation concerns, especially for the set-up of management strategies and the definition of distinct evolutionary significant units.
authors.affiliation: From the Unité Évolution, Écologie et Paléontologie, UMR 8198 CNRS/Université de Lille-Sciences et Technologies, F-59655 Villeneuve d'Ascq cedex, France.
authors.forename: Leslie
authors.initials: L
authors.lastname: Faucher
authors.affiliation: From the Unité Évolution, Écologie et Paléontologie, UMR 8198 CNRS/Université de Lille-Sciences et Technologies, F-59655 Villeneuve d'Ascq cedex, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Godé
authors.affiliation: From the Unité Évolution, Écologie et Paléontologie, UMR 8198 CNRS/Université de Lille-Sciences et Technologies, F-59655 Villeneuve d'Ascq cedex, France. jean-francois.arnaud@univ-lille1.fr.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Arnaud
mesh_terms: D000483:Alleles; D000818:Animals; D002025:Bufonidae / Q000145:classification* / Q000235:genetics*; D004272:DNA, Mitochondrial; D014644:Genetic Variation; D005828:Genetics, Population; D005838:Genotype; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D004272:DNA, Mitochondrial
keywords: 
  Bufo calamita
  ; Kompetitive allele-specific PCR; amphibians; conservation genetics; genetic diversity; mtDNA SNPs; nuclear microsatellites; parentage