Data from: 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)

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 five 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 mtDNA 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.全球范围内两栖动物正经历大规模种群衰退，该类群受威胁物种数量持续攀升，凸显了利用高质量分子标记开展保护遗传学研究的迫切需求。黄条蟾蜍（Bufo (Epidalea) calamita）是一种易危的拓殖性物种，仅分布于西欧的特化生境中。为向保护生物学家提供高效且经济的遗传资源，本研究借助下一代测序技术开发并验证了22对全新的核微卫星标记。本研究同时利用桑格（Sanger）测序获得的序列数据，开发了首个适用于KASPar分型检测的线粒体标记。本研究对采自法国5个种群的95只黄条蟾蜍进行了遗传多态性分析。对于多态性微卫星位点，其等位基因数与期望杂合度分别介于2~14与0.035~0.720之间。未检测到显著偏离随机交配的情况（多位点平均FIS=-0.015），且种群间分化程度显著（多位点平均FST=0.222，P<0.001）。针对16S与D-loop区域的18个线粒体单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点，本研究进一步开发了基于竞争性等位基因特异性PCR扩增（KASPar）的快速、经济的SNP分型方法。这些线粒体SNP标记的等位基因组合共产生10种不同的单倍型，种群内单倍型数量介于2~5之间。种群间分化程度较高（GST=0.407，P<0.001）。本研究开发的全新遗传资源将为后续的亲权鉴定、种群遗传学以及系统地理学研究提供助力，同时可应用于进化与保护相关研究，尤其有助于制定种群管理策略以及界定独立的进化显著单元（Evolutionarily Significant Unit, ESU）。