From our list of putative SNPs across the C. vestalis genome, we selected 100 SNPs for genotyping assay development. We first selected the 200 largest scaffolds; they varied in length from 17-58Kb and contained a total of 7,878 SNPs. We then removed SNPs with a minor allele frequency (MAF) <0.2, SNPs that had another SNP within 50 bp up- or downstream, and SNPs with more than 2 alleles. The remaining SNPs were binned in MAF bins of 0.2-0.3 (1,908 SNPs), 0.3-0.4 (1,605 SNPs) and 0.4-0.5 (1,156 SNPs). Per MAF bin, SNPs were ranked by SNP quality score. We then selected the SNPs with the highest quality scores, picking 20 SNPs with a MAF between 0.2-0.3 and 40 each with a MAF between 0.3-0.4 and 0.4-0.5, all on different scaffolds. All selected SNPs had a quality score of more than 200 (based on SAMTOOLS), and an average read depth of 61. High-throughput genotyping assays based on allele-specific forward primers were developed for these 100 SNP sequences at KBioscience (now LGC Genomics, Hoddesdon, U.K.)
