섞여있는 회유성 어류 계군을 그 기원에 따라 어떻게 구분할 것인가?: 단염기다형성을 이용한 단순하고 빠른 PCR 방법

Abstract

Migratory fishes often intermix in the open ocean among stocks of different origins sometime during their life. The chum salmon (Oncorhynchus keta), an anadromous fish that distributes in the North Pacific is one of such examples. The fish reproduces in the river, migrates into the open ocean for growth and becomes intermixed. To study stock-specific migratory routes and other ecological characteristics, it will be necessary to develop a method discriminating each stock. Here, we present a simple polymerase chain reaction (PCR) method utilizing single nucleotide polymorphisms (SNPs) observed in the COIII-ND3-ND4L region of the mitochondrial DNA. From a 744 nucleotide-long sequence of the region, several stock-specific SNPs were observed (e.g., positions 57, 70, 246, 303, 307, 534, 591) among 141 chum salmons of Korea, Japan, Canada, and the United States. Based on these SNPs, 20-25 nucleotide-long stock-specific PCR primers were designed in a way that the most 3'end nucleotide becomes identical to the SNPs and that the second to the 3'end mismatches the conserved nucleotide. With primer pairs designed as such, PCR amplified DNA distinctively among the Korea, Japan, Canada and the United States chum salmons: e.g. primer pairs of SF0 and SR1 for Korea and of SF1 and SR2 for America salmons. Because of its simplicity, this method would be useful for processing many specimens of aggregated stocks collected during any open ocean sampling cruise.