Identification of S-genotypes by PCR-RFLP in breeding lines of Brassica

We developed a molecular method for the identification of the S-alleles of Brassicaceae, which belongs to the inbred line. This method is quicker and more precise than the existing methods. The genotype of the S-allele for 20 S-haplotypes of cabbage and 20 S-haplotypes of broccoli was determined by a pollination test. In order to identify the S-alleles, we performed PCR-RF LP with a mixture of the primers that are related to the S-locus glycoprote in (SLG) gene, which corresponds to the results of the pollination test. Th e selected primers amplified all of the single bands of about 1,150 bp in a ll 40 lines of cabbage and broccoli. Three out of 20 lines of cabbage were amplified by class I SLG specific primers, whereas all of the lines of the cabbage were amplified by class II SLG specific primers. Therefore, we coul d not classify class I and claps II precisely by the class I and class II p rimers. However, 15 out of 20 lines of broccoli were amplified by the class I SLG specific primers. The remaining 5 lines were amplified with the clas s II SLG specific primers. We then digested the amplified PCR products with various restriction endonucleases and chose a restriction endonuclease, wh ich accords exactly with the results of the diallel cross. The best one was HinfI. Its RFLP result was the same as that of the nucleotide sequence ana lysis. The 40 lines of cabbage and broccoli consisted of 16 different S-hap lotypes. Therefore, the PCR-RFLP analysis was quicker and more precise in i dentifying the characteristics of S-haplotypes that are used in breeding. A lso, we were able to check whether the lines could be mixed. The S-genotype s were difficult to determine due to the different flowering time.