The conversion of a RFLP assay into PCR for the determination of purity in a hybrid pepper cultivar

A RFLP assay which had been developed for distinguishing between a hybrid pepper and its parents, has been converted into a simple applicable PCR assay, suitable for large-scale quality-control assessment of the commercial hybrid. For this conversion the sequences of the ends of the probe used in the previous RFLP assay were determined. From these sequences suitable primers were devised for inverse PCR of heterogeneous DNA fragments derived from the male parent. The inverse-PCR product was cloned and partially sequenced. These sequences, in turn, made it possible to determine primers on both sides of the locus of mutation, and to develop the reported conventional PCR assay.