2.6 PCR-RFPL analysis
17 µl of the PCR amplicons were digested using the respective restriction enzymes (AccI, AciI, AleI, AlwNI, ApoI, AvaI, BbvI, BccI, BceAI, BclI, BsaAI, BsaBI, BsaI, BsiEI, BsmI, BspMI, BsrI, BstAPI, HaeII, HaeIII, HgaI, HinfI, HpaII, HphI, NcoI, NspI, PleI, PstI, PvuII, RsaI, Tth111I, XcmI; all from New England Biolabs) and recommended concentrations. PCR products were digested for 4h or overnight to avoid partial digestion and loaded on a 1% agarose gel. Genotypes were determined by visual inspection of gels and gel photographs (Fig. S3 and S4). Sometimes, despite long digestion time, we observed incomplete digestion. Genotypes were assigned as follows: 1) A lack of a smaller fragment was always interpreted as a homozygous genotype. 2) A band at the position of the uncut amplicon together with a second band was interpreted as a heterozygous genotype, but only if the uncut band was stronger in intensity (as the same number of longer-sized DNA molecules results in a stronger signal). 3) Complete lack of a band at the size of the uncut fragment or a band that had less intensity than the digested fragments were interpreted as a homozygous genotype for the alternative allele.