Kurata Seikan, Mano Shota, Nakahama Naoyuki, Hirota Shun K, Suyama Yoshihisa, Ito Motomi
Tomakomai Experimental Forest, Hokkaido University, Tomakomai, Japan Tomakomai Experimental Forest, Hokkaido University Tomakomai Japan.
Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan Faculty of Bioresource Sciences, Akita Prefectural University Akita Japan.
Biodivers Data J. 2024 Jun 18;12:e117014. doi: 10.3897/BDJ.12.e117014. eCollection 2024.
Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.
昆虫是地球上最多样化的真核生物群体之一，现存物种达100万种或更多，包括尚未描述的物种。DNA条形码系统已得到发展，它有助于识别隐存物种和未描述物种。线粒体细胞色素氧化酶I区域（mtDNA COI）已被用于昆虫类群的条形码分析。此后，下一代测序（NGS）技术得到发展，使得能够快速获取大量序列数据用于遗传分析。尽管已经开发出基于NGS的用于扩增mtDNA COI区域的PCR引物，但其靶区域只是COI区域的一部分，和/或存在PCR扩增的分类学偏差。由于mtDNA COI区域是DNA条形码系统的传统DNA标记，针对该区域的改良引物将极大地有助于分类学研究。在本研究中，我们重新设计了先前开发的靶向mtDNA COI条形码区域的PCR引物组，以提高扩增效率，并使我们能够对NGS进行测序分析。结果，重新设计的引物组在本研究中所检测的物种中成功率很高（> 85%），涵盖四个昆虫目（鞘翅目、鳞翅目、直翅目和蜻蜓目）。因此，通过将这些引物与已开发的用于12S或16S rRNA区域的引物组相结合，我们可以使用NGS进行更详细的分类学、系统地理学和保护遗传学研究。
