An F2 population was developed from a cross between a mur-cytoplasmic male sterile broccoli line and a restorer Chinese kale line. Phenotypic analysis of F2 plants indicated that the pollen fertility is controlled by two genes and segregated in a duplicate gene interaction mode with a ratio of 15:1. A total of 236 single nucleotide polymorphism (SNP) markers were developed utilizing 1,448 primers designed for production of expressed sequence tag (EST)-SNP markers of Raphanus sativus and analyzed by the dot-blot technique in 205 F2 individuals. A linkage map was constructed with a total of 142 markers and these markers were assigned to nine linkage groups together with simple sequence repeat markers mapped previously on the published linkage maps of Brassica oleracea. The linkage map spanned 909 cM with an average marker distance of 6.4 cM. A fertility restorer locus (Rfm1) was mapped on LG1, corresponding to chromosome 3, along with a flower color locus at a distance of 25 cM. SNP markers flanking the Rfm1 locus were BoCL2642s at a distance of 2.5 cM on one side and BoCL2901s at a distance of 7.5 cM on the other side. All the SNP markers showed homology with Arabidopsis thaliana and Brassica rapa genome sequences. Three pentatricopeptide repeat genes of the P-subfamily, particularly expressed in buds of the restorer line, were identified and these genes could be potential candidate fertility restorer genes.
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