Schematic overview of the polymorphism discovery strategy. PCR on genomic DNA of 96 testing strains and on a set of 96 samples of the reference strain Brown Norway (BN/Cl) produces two sets of 96 PCR products that contain different fluorescent labels at either end (IRD700 and IRD800, indicated by red and blue stars). Pooling of tester samples with reference samples, followed by denaturing and reannealing, results in the formation of heteroduplex DNA in the case of polymorphisms between the strains used. Heteroduplex DNA is specifically cleaved by the CEL I nuclease at the site of the mismatch and the resulting fragments are separated on a denaturing polyacrylamide gel with fluorescence detection. Subsequently, representative samples for a specific polymorphism are sequenced to reveal the molecular nature of the polymorphism.
