FIGURE 2 Workflow of the whole genome re-sequencing-based
design of PCR-RFLP markers (GB-RFLP). A. Markers were designed based on
genetic differentiation (FST) or genome-wide
genotype-phenotype association (GWA) data of a 453-genome dataset using
pairwise ingroup–outgroup comparisons. B. Variants were screened for
RFLPs with one allele (but not the other) being cut by a restriction
enzyme. C. Target variants were filtered based on the presence of
additional restriction sites and additional SNPs within the restriction
site. Quality control was performed by plotting allele frequencies and
haplotype networks. D. Likelihoods that a genotype corresponds to a
population (or not) were calculated based on population-specific allele
frequencies. Percentage of correct assignments together with false
negative and false positive rates were based on bootstrapping of
genotypes (informed by empirical allele frequencies in the genomic
dataset). E. Primers were designed based on 801 bp sequences (core SNP
+/- 400 bp) in a way that the restriction enzyme would generate two
fragments with a ~1:2 length ratio. F. PCR conditions
were tested and optimized. G. Restriction digest was performed on 8
ingroup samples and 5–8 outgroup samples. Genotypes were determined by
visual inspection. These data were used to calculate the number of
correct assignments as well as rates of false positives and negatives
(as for the bootstrapping dataset in D).