Species Identification of Pacific Salmon by Means of a Major Histocompatibility Complex Gene

—A rapid genetic test to identify Pacific salmonid tissue samples to the species level is described. An exon (coding DNA) and its adjacent intron (noncoding DNA) of a major histocompatibility complex (MHC) class II gene were amplified by the polymerase chain reaction from eight Oncorhynchus species and the two Salmo species that have been transplanted to British Columbia. Among Pacific salmonids, the length of the amplified sequence was between 809 and 826 base pairs (bp) for cutthroat trout Oncorhynchus clarki. rainbow trout and steelhead O. mykiss. chinook salmon O. tshawytscha. coho salmon O. kisutch, masu O. mason, and some sockeyc salmon O. nerka: it was between 993 and 1,034 bp for pink salmon O. gorbuscha, chum salmon O. keta. and other sockeyc salmon. Sequence length ranged from 1,000 to 3,000 bp for brown trout Salmo trutta and from 1.500 to 3,000 bp for Atlantic salmon S. salar. Amplified sequences from all Pacific salmonids except rainbow trout-steelhead and cutthroat trout displayed species-specific restriction fragment length polymorphisms (RFLPs) after independent digestion with three restriction enzymes (Avrll. Bell. B s o f l ) . Restriction patterns of Pacific salmon sequences between 993 and 1,034 bp distinguished them from the 1,000-bp brown trout sequences. Intraspecific RFLP variability revealed regional differentiation in phenotypic frequencies in three species: coho salmon populations in southern British Columbia differed from those in northern British Columbia and the Frascr River; sockeye salmon from Kamchatka and Bristol Bay differed from those of British Columbia; and Japanese and North American chum salmon were well differentiated, enabling an accurate classification to continent of origin. Correct species identification is of fundamental importance in the management and conservation of Pacific salmon Oncorhynchus spp. Identification of species is routine when the fish are approaching sexual maturity, given the distinctive physical changes that occur. However, species identification can be more problematical when juveniles are to be identified, even for experienced observers. In some cases, species identification is required when only a portion of a fish (e.g., a fillet) is available after having been processed and possibly cooked, or when a sample has been preserved in formalin or ethanol. Species identification of fish independent of morphological characteristics has centered on the use of genetic and immunological variation. When adequate tissue samples of suitable quality arc available, electrophoretic analysis of isozymes can be an effective method of species discrimination (Mork el al. 1983; Keenan and Shaklee 1985; Graves et al. 1989). However, when there is an inadequate amount of tissue for analysis or the tissue is of poor quality, amplification of selected DNA fragments via polymerase chain reaction (PCR) may be required (Pendas et al. 1995; Unseld et al. 1995). Species identification of tunas Thunnus spp. was accomplished initially by direct sequencing of an amplified mitochondrial DNA fragment (Bartlett and Davidson 1991). Subsequently, analysis of restriction fragment length polymorphisms (RFLPs) of a longer mitochondrial fragment was developed for tuna species identification (Chow and Inoue 1993), enabling relatively simple laboratory analysis. The PCR amplification of a DNA sequence and subsequent RFLP analysis of the fragment has also been applied to a number of other species identification problems (Silberman and Walsh 1992; Chow et al. 1993). Substantial species differences have been reported in the nucleotide sequence of a major histocompatibility complex (MHC) class II pi gene in Atlantic salmon Salmo salar and Pacific salmon (Hordvik et al. 1993; Grimholl et al. 1994; Miller and Withler 1996). The MHC genes are involved in the function of the immune system; T cells recognize a foreign antigen only in association with class I or II molecules encoded by the MHC. The MHC class II gene sequenced in Atlantic and Pacific salmon displays both intraand interspecific polymorphism. Species-specific DNA sequences occur in both the DNA sequences that encode proteins (exons) and the interspersed noncoding DNA sequences (introns). In this study, we developed techniques to amplify a contiguous MHC class II