, Volume 6, Issue 2, pp 133-138

Development of a method for the identification of S alleles in Brassica oleracea based on digestion of PCR-amplified DNA with restriction endonucleases

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The development of a technique for the identification of S alleles involved in self-incompatibility in Brassica oleracea which is based on the polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction analysis is described. Primers homologous to conserved regions near to the 5′ and 3′ ends of the S coding sequence were used to amplify a number of members of the S multigene family. However, by designing a selective primer and using a higher temperature for annealing in the PCR, we were able to amplify certain members from the multigene family preferentially. These were considered to be the S-locus glycoprotein genes (SLG), since the patterns of restriction bands of the PCR products were shown to correspond to those of the SLG where sequence data were available. DNA samples from plants with certain S alleles were found not to amplify efficiently using the “selective” primers and high annealing temperature. This property, however, could be used as a means of distinguishing plants homozygous for these S alleles, as was demonstrated by an examination of a small F2 population that was segregating for the S5 and S29 alleles. In the investigation of the F2 population, it was found that preferential amplification of one of the alleles of the heterozygotes occurred when the “consensus” primers were used in the PCR. However, by using different primers, homologous to another region of the S sequence, we were able to amplify both alleles of the heterozygotes equally. The genotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen-tube growth tests.