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.