Figure 5

RAD Amp: a PCR-based method for genotyping individual RAD markers. (a) Genomic DNA is digested, ligated to short linkers (orange bar), and used as polymerase chain reaction (PCR) template for restriction site associated DNA (RAD) Amp. (b) RAD Amp primer design takes advantage of primer-template mismatching to discourage aberrant extension in the absence of linker. In the AB sample, the polymorphism disrupting the restriction site is red. (c) RAD Amp was performed using primers specific to the seven RAD markers linked to b1166. Amplification using these primers with WIK and AB template showed WIK-specific allele detection. RAD Amp detected WIK alleles in a pool containing 0.5% WIK and 99.5% AB DNA. WIK alleles, from the wild-type parent, were detected in a pool of 24 F₂ mutant individuals from the b1166 mapping cross at WIK18I13, WIK07C02, WIK05E18, and WIK13P08. No WIK alleles were detected at WIK20J21, WIK18C01, or WIK11I12, suggesting that these markers are the more closely linked to the mutation.