New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla
New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla
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DOI：
10.1038/s41598-019-44402-0
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发表时间：
2019-05-28
影响因子：
4.6
通讯作者：
Miya, Masaki
中科院分区：
文献类型：
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作者：
Takeuchi, Aya;Sado, Tetsuya;Miya, Masaki
淡水鳗鱼属的鳗鱼包括16个物种，其中包括三个亚种，其特点是其独特的降河产卵的生命周期。它们的生活史和夜间生活方式使它们很难在淡水和海洋栖息地观察到。为了研究它们在水生环境中的分布和生态，我们开发了一种新的PCR引物，从安圭拉环境DNA（eDNA）的元条形码。新的引物（MiEel）是针对线粒体ATP 6基因的两个保守区域设计的，它扩增了一个可变区，该可变区具有足够的种间变异，范围从5到22个核苷酸差异（3个亚种对之间的1到3个核苷酸差异）。我们证实了多功能性的MiEel引物的所有淡水鳗鱼组织DNA提取物时，分别分析。使用模拟社区与MiEel引物相结合的metabarcoding能够在物种水平上同时检测安圭拉。对来自水族箱、受控池塘和天然河流的eDNA样本的分析表明，MiEel元条形码可以成功地从水样中检测出正确的安圭拉物种。这些结果表明，利用MiEel引物构建的eDNA元条形码可用于非侵入性监测难以进行抽样调查的水生环境中濒危鳗鲡的存在。
Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.