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利用下一代测序技术和竞争性等位基因特异性PCR(KASPar)开发黄条蟾蜍(Bufo (Epidalea) calamita,蟾蜍科)的核微卫星位点和线粒体单核苷酸多态性

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).

作者信息

Faucher Leslie, Godé Cécile, Arnaud Jean-François

机构信息

From the Unité Évolution, Écologie et Paléontologie, UMR 8198 CNRS/Université de Lille-Sciences et Technologies, F-59655 Villeneuve d'Ascq cedex, France.

出版信息

J Hered. 2016;107(7):660-665. doi: 10.1093/jhered/esw068. Epub 2016 Sep 15.

DOI:10.1093/jhered/esw068
PMID:27634537
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 F = -0.015) and population differentiation was substantial (mean multilocus F = 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 (G = 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.

摘要

两栖动物在全球范围内正经历着重大衰退,这一特定分类群中受威胁物种数量的稳步增加凸显了使用高质量分子标记进行保护遗传学研究的必要性。黄条蟾蜍(Bufo (Epidalea) calamita)是一种易危的先锋物种,局限于西欧的特殊栖息地。为了为保护生物学家提供高效且经济高效的遗传资源,我们利用下一代测序技术开发并鉴定了22个新的核微卫星标记。我们还使用从桑格测序获得的序列数据开发了用于KASPar分析基因分型的首个线粒体标记。然后对从法国5个种群中采集的95只蟾蜍进行了遗传多态性分析。对于多态微卫星位点,等位基因数量和预期杂合度分别为2至14个以及0.035至0.720。未观察到明显偏离随机交配的情况(平均多位点F = -0.015),且种群分化显著(平均多位点F = 0.222,P < 0.001)。从位于16S和D环区域的一组18个线粒体单核苷酸多态性(SNP)中,我们进一步开发了一种基于竞争性等位基因特异性PCR扩增(KASPar)的快速且经济高效的SNP基因分型方法。这些线粒体DNA SNP标记的等位基因状态组合产生了10种不同的单倍型,每个种群内的单倍型数量为2至5种。种群间差异高度显著(G = 0.407,P < 0.001)。这些新的遗传资源将有助于未来的亲权鉴定、种群遗传学和系统地理学研究,并且对于进化和保护问题都将是有用的,特别是对于管理策略的制定以及不同进化显著单元的定义。

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