MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.
MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.
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DOI：
10.1098/rsos.150088
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发表时间：
2015-07
影响因子：
3.5
通讯作者：
Iwasaki W
中科院分区：
文献类型：
--
作者：
Miya M;Sato Y;Fukunaga T;Sado T;Poulsen JY;Sato K;Minamoto T;Yamamoto S;Yamanaka H;Araki H;Kondoh M;Iwasaki W
我们开发了一套用于编码鱼类环境DNA(EDNA)的通用聚合酶链式反应引物(MiFish-U/E)。用来自880个物种的线粒体全基因组(有丝分裂组)序列设计了引物，并补充了来自160个鳍科动物(鲨鱼和鱼类)的部分有丝分裂组序列。该引物针对12SRRNA基因的一个高变区(163185rRNA碱基)，除了一些密切相关的同系物外，包含了足够的信息来识别鱼类的分类科、属和种。为了测试这些引物在多种鱼类中的通用性，我们从冲绳久久美水族馆的四个已知物种组成的水族箱中采集了EDNA样本，准备了双索引文库，并使用高通量下一代测序技术对该区域进行了配对末端测序。在4个水族箱中包含的180种海洋鱼类中，我们检测到168种(93.3%)，分布于59科123属。这些鱼不仅在分类上有多样性，从鲨鱼和射线到高等硬骨鱼，而且它们的生态也有很大的变化，包括生活在浅海到深海的中上层和底栖生物物种。我们还对水族馆附近的珊瑚礁周围的天然海水进行了采样，并使用这种方法检测到了93种鱼类。在93种水族箱中，4个水族箱中未检出，共检出物种232种(隶属于70科152属)。与传统的调查方法相比，本文提出的元编码方法是一种非侵入性的、更有效、更具成本效益和更敏感的方法。它有可能作为生物多样性监测的替代(或补充)工具，在更大的空间和时间尺度上彻底改变自然资源管理和鱼类群落的生态研究。
We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163–185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.