Development of 5’-Nuclease Reactions for High-Throughput SNP Genotyping in Salmon

Information on the oceanic migration patterns and relative marine survival of individual stocks is critical to our understanding of fluctuations of salmonid populations under changing climatic conditions. Migrations following stock-specific corridors may lead to differing marine survival and varying rates of return among stocks during periods of changing marine conditions. By comparing genetic attributes of collections of fish taken in high-seas and near-shore areas with those characteristic of potentially contributing stocks one can infer the origin of the collection and thus a point on the migratory route of that stock. Single nucleotide polymorphisms (SNPs) are a class of genetic markers consisting of differences in DNA bases between individuals or individual chromosomes. These polymorphisms have been assayed in salmonids and other taxa using a wide variety of technologies over the past couple of decades. Although many of these SNPs provided powerful information for fisheries management, the technologies used to collect genotype data for them were slow and expensive relative to those for alternative marker classes (e.g. allozymes and microsatellites). In recent years several high-throughput, low-cost SNP genotyping techniques have been developed (several are described in Kwok 2003). We are developing markers based on one of these techniques known as the 5’-nuclease reaction (Fig. 1) and are using these markers to genotype SNPs in large numbers of chinook, sockeye and chum salmon. These SNP genotyping assays are being developed in order to utilize the wealth of previously described polymorphisms that have not been widely applied to migration or mixture studies due to throughput constraints of older technologies. Using two thermal cycler blocks four times per day, we observed that a single technician could genotype over 3,000 individuals. Since thermal cycling was the limiting step, the use of additional cycler units could multiply this throughput rate several times. The relative simplicity of the raw data analysis (Fig. 2) and the lack of an electrophoresis component rendered genotyping based on the 5’nuclease reaction faster and thus less expensive than genotyping using the techniques originally published for each SNP. This greatly increases the potential utility of SNPs for fishery management applications. Fig. 1. Genotyping via the 5’-nuclease reaction involves adding allele-specific fluorescent oligonucleotide probes to a typical polymerase chain reaction. During the reaction the probes anneal to the template DNA either (1a) perfectly or (1b) imperfectly. In the former case, the probe is cleaved causing fluorescence. In the latter case, the probe is simply knocked free and no color change is observed.