Identification of Novel Single-Nucleotide Polymorphisms in Chinook Salmon and Variation among Life History Types

—Single-nucleotide polymorphisms (SNPs) are simple base substitutions or small indels in otherwise conserved regions of nuclear or mitochondrial DNA. Assays detecting these genetic markers have been a powerful tool for conserving and managing fish populations. In nonmodel species, however, available DNA sequence data are limited and inventive techniques must be employed in screening for SNPs. In this study, we used expressed sequence tags from rainbow trout Oncorhynchus mykiss to design primers for amplification of genomic DNA fragments from Chinook salmon O. tshawytscha. The regions flanking the repeat sequence of published microsatellite loci in Chinook salmon were also chosen to examine nucleotide variation. Thirty-two individuals representing the majority of the species’ range from Russia to California were sequenced at 19 loci to identify SNPs. We discovered 58 polymorphisms during the screen and chose 13 for development into 5 exonuclease assays (TaqMan assays). The genotypes from the TaqMan assays were compared with sequence data from individuals from the ascertainment panel to confirm proper allele designations. To evaluate the power of these markers to differentiate populations and life history types, we typed 91 individuals representing the major lineages of Chinook salmon with 11 validated assays. Significant allele frequency differences were observed among life history types of Chinook salmon at seven SNP loci, demonstrating the power of these markers to differentiate lineages. Chinook salmon Oncorhynchus tshawytscha are the largest of the seven species of Pacific salmon. They spawn in drainages of the North Pacific Ocean in both North America and Asia. Several populations of this species have vanished over the past century due to human influence on spawning habitat (Nehlsen et al. 1991). Their economic importance to the fishing industry and cultural significance to indigenous peoples has given rise to extensive conservation efforts. Such efforts include hatchery supplementation, harvest management, and habitat improvement. Conservation genetics research allows evaluation of the effects of management efforts on populations and provides information to guide future decisions. The two most distinct forms of Chinook salmon, known as the ocean and stream types, are characterized by differences in juvenile out-migration, adult run timing, and spawning location. Ocean-type Chinook salmon typically have a brief juvenile period in freshwater before migrating to the ocean as subyearlings and return as adults in summer and fall to spawn in relatively warm main-stem rivers (hence, they are also called ‘‘summer run’’ and ‘‘fall run’’; Healey 1991). Stream-type Chinook salmon have a prolonged juvenile phase in freshwater and out-migrate as yearlings; the adults tend to make longer spawning migrations to small, cold tributaries in the spring (hence, they are also termed ‘‘spring run’’; Healey 1991). Population genetics studies in Pacific salmon, such as those related to population differentiation, parentage analysis, and genetic stock identification, rely on a panel of genetic markers. Such markers have included allozymes, microsatellites, mitochondrial DNA (mtDNA), and single-nucleotide polymorphisms (SNPs). In recent years, the most commonly used genetic marker for Pacific salmon has been microsatellites (Bentzen et al. 1991; Nelson et al. 1998; Nelson et al. 2001). The use of microsatellites over roughly the last 10 years has illuminated fine-scale population structure among Chinook salmon in numerous regions and helped shape fisheries management programs (Beacham et al. 2006). However, combining or sharing genotype data between laboratories requires costly and arduous standardization and maintenance efforts (Seeb et al. 2007). These technical issues and the potential for reduced costs have prompted many researchers to explore alternative types of genetic markers such as SNPs (Smith et al. 2005a). Single-nucleotide polymorphisms are defined simply as single base substitutions in a sequence of DNA. A looser definition of the term, however, also includes small deletion and insertion polymorphisms (see the National Center for Biotechnology Information’s Web site, www.ncbi.nlm.nih.gov/projects/SNP/). Reported to occur approximately once in every 300 base pairs for Chinook salmon (Smith et al. 2005a), SNPs are the most common type of genetic sequence variation (Morin et al. 2004). Screening for SNPs in salmonid species has become attractive in recent years because of a rapidly growing collection of expressed sequence * Corresponding author: [email protected] Received January 11, 2007; accepted July 27, 2007 Published online January 14, 2008 96 Transactions of the American Fisheries Society 137:96–106, 2008 Copyright by the American Fisheries Society 2008 DOI: 10.1577/T07-011.1 [Note]