Development of the Restriction Site Tiling Analysis (RSTA) method: Accurate discovery and quantitative genotyping of genome-wide polymorphisms using nucleotide arrays

Here we developed a new high-throughput polymorphism detection and genotyping method based on identifying restriction cut site polymorphisms using a microarray platform. We compared the genomes of 20 individual urchins; 10 from the northern part of the species range (Boiler Bay, OR) and 10 from the southern part of the range (San Diego, CA). Genomic DNA from each individual was extracted, randomly sheared, then divided in half. Half was restriction digested and labeled with Cy3, half was treated as control and labeled with Cy5. Labeled DNA from a single individual was then competitively hybridized to the custom, species-specific array that had probe sequences designed to be centered on known restriction cut sites. Resulting log ratio data reflected the three possible genotypes in clusters when arrays from multiple individuals were examined at a given polymorphic locus.