DEVELOPMENT OF A METHOD FOR THE IDENTIFICATION OF S-ALLELES IN BRASSICA-OLERACEA BASED ON DIGESTION OF PCR-AMPLIFIED DNA WITH RESTRICTION ENDONUCLEASES

The development of a technique for the identification of S alleles inv olved in self-incompatibility in Brassica oleracea which is based on t he polymerase chain reaction (PCR) amplification of genomic DNA follow ed by restriction analysis is described. Primers homologous to conserv ed regions near to the 5' and 3' ends of the S coding sequence were us ed to amplify a number of members of the S multigene family. However, by designing a selective primer and using a higher temperature for ann ealing in the PCR, we were able to amplify certain members from the mu ltigene 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 se quence data were available. DNA samples from plants with certain S all eles were found not to amplify efficiently using the ''selective'' pri mers and high annealing temperature. This property, however, could be used as a means of distinguishing plants homozygous for these S allele s, as was demonstrated by an examination of a small F2 population that was segregating for the S5 and S29 alleles. In the investigation of t he F2 population, it was found that preferential amplification of one of the alleles of the heterozygotes occurred when the ''consensus'' pr imers were used in the PCR. However, by using different primers, homol ogous to another region of the S sequence, we were able to amplify bot h alleles of the heterozygotes equally. The genotypes of the plants we re determined by restriction analysis of PCR products and agreed with results based on pollen-tube growth tests.