Semiautomated multilocus genotyping of Pacific salmon (Oncorhnychus spp.) using microsatellites.

We report the development of a semiautomated multilocus genotyping system for Pacific salmon using four-color fluorescent detection of microsatellites. An initial screening of microsatellites was conducted on five species of Pacific salmon (Oncorhynchus spp.) and Atlantic salmon (Salmo salar) using 35 primer pairs developed from six species of salmonid. The number of loci that amplified varied by species from 11 (chum salmon) to 22 (chinook salmon). We then tested co-amplification of microsatellites in chinook, coho, and sockeye salmon and developed six-locus multiplex systems. The species-specific multiplex systems were applied to two populations using a sequencer/gene scanner (Perkin-Elmer Applied Biosystems, Inc. [ABI] 373A). The genetic variability at each locus was calculated to evaluate the utility of this system for genetic studies. Significant differences in allele frequencies were observed between populations in 14 of 18 pair-wise comparisons. Average heterozygosity ranged from 0.47 in Togiak River coho salmon to 0.75 in Dungeness River chinook salmon. Observed heterozygosities ranged from 0 at One mu 1 in Togiak River coho to 0.96 at Ssa85 in Dungeness River chinook. The probability of match (PM) for each six-locus multiplex system was 4.0 x 10(-10), 7.2 x 10(-8), and 3.2 x 10(-7) for chinook, coho, and sockeye, respectively. The average polymorphic information content (PIC) was 0.77, 0.56, and 0.60 for chinook, coho, and sockeye, respectively. The microsatellite loci used here show promise for high-resolution genetic studies of Pacific salmon such as fine-scale population analysis kinship, and parentage studies.