RESUMO
Riverine metabarcoding data were obtained from the Takamigawa River, a tributary of the Kinokawa River, in Nara Prefecture (Central Honshu, Japan). We extracted DNA from bulk community samples of aquatic organisms, most of which could not be morphologically identified at species level due to their small body size (0.12 - 2 mm length). A partial coding region of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified using PCR, and the amplicon was subjected to high-throughput parallel sequencing (Illumina MiSeq). The 313 bp paired-end sequence reads were classified into operational taxonomic units (OTUs), their species boundaries were delineated using the Generalised Mixed Yule Coalescent (GMYC) method, and taxonomic names of the GMYC species were assigned using basic local alignment search tool (BLAST) against International DNA Databases (INSD: GenBank, ENA, and DDBJ).
RESUMO
We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera, Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences of histone H3 and EF-1alpha genes are determined for some insects with PCR-amplified DNA fragments.
