Mitochondrial DNA sequence divergence between “Kunimasu” Oncorhynchus kawamurae and “Himemasu” O. nerka in Lake Saiko, Yamanashi Prefecture, Japan, and their identification using multiplex haplotype-specific PCR

Abstract

In order to develop a simplified method for discriminating between Oncorhynchus kawamurae (Kunimasu) and sympatric Oncorhynchus nerka (Himemasu), sequence variations of mitochondrial DNA control region were examined on 71 specimens of Kunimasu and 103 of Himemasu. Sequence analyses for 530 bp revealed 3 variable sites, which defined three haplotypes in Kunimasu and one in Himemasu. Based on determined nucleotide sequences, species-specific primers were designed for the multiplex haplotype-specific polymerase chain reaction, which amplified ca. 900 and 1,040 bp fragments in Kunimasu and Himemasu, respectively. This simple rapid approach can be used to distinguish between the two species without the need to sacrifice specimens.