Development of novel PCR primer sets for DNA metabarcoding of aquatic insects, and the discovery of some cryptic species

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification by using standardized genetic region(s). It can be a powerful tool in various fields of biology such as for revealing the existence of cryptic species and/or rare species and in environmental science such as when monitoring river biota. Biodiversity reduction in recent times has become one of the most serious environmental issues on a worldwide scale. DNA barcoding techniques require the development of sets of universal PCR primers for DNA metabarcoding. We tried to develop universal primer sets for the DNA barcoding of all insect groups. In this study, we succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region. A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. Such resulting database enhancements will serve as a powerful tool for increasingly accurate assessment of biodiversity and genetic diversity.