Multiplexed systems of microsatellites for genetic analysis in coastal cutthroat trout (Oncorhynchus clarki clarki) and steelhead (Oncorhynchus mykiss)

We report the first application of microsatellites to genetic surveys of the endangered coastal cutthroat trout (Oncorhynchus clarki clarki). We also describe the application of microsatellites derived from other species to steelhead/rainbow trout (O. mykiss), thus extending the suite of microsatellite markers available for this species. We screened 35 microsatellite primer sets previously developed from six salmonid species to assess their utility as genetic markers for cutthroat and steelhead; of these, 21 primer sets yielded high-quality amplification products in both species. We used 10 primer sets to develop four species-specific multiplex sets, containing six and three loci for cutthroat, and six and four loci for steelhead, and applied them to three populations of each species. Each of the four sets was amplified in a single polymerase chain reaction (PCR) using fluorescently labeled primers, visualized in a single gel lane, and analyzed rising a semiautomated multilocus genotyping system. Numbers of alleles per locus averaged 12 in cutthroat and 8 in steelhead populations. Observed heterozygosities were 0.26-0.97 in cutthroat and 0.00-0.92 in steelhead (mean = 0.70 and 0.54, respectively). Significant differences in allele frequencies were found in 54 of 57 pairwise interpopulation comparisons in the two species. The probabilities of match (P(M)) for the 9-locus and 10-locus systems were very low in each of the three populations surveyed in cutthroat and steelhead (1.2 x 10^-16 to 4.5 x 10^-15 and 4.2 x 10^-15 to 6.2 x 10^-8 respectively). In all but true population, the average polymorphic information content (PIC) per locus was greater than 0.5, and therefore considered highly informative. These multiplex methods are reproducible, resolve, extensive genetic variation, and are compatible with nonlethal sampling; hence, they should prove useful for a wide range of genetic investigations involving coastal cutthroat trout and steelhead.