Development of molecular markers specific to petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee).

To establish a simple and rapid method for isolating mitochondrial DNA (mtDNA) from Brassica vegetables, the effects of different factors on mtDNA extraction were investigated firstly. A new protocol includes five steps: organelle isolation, deoxyribonuclease treatment, lysis, RNase treatment, and deproteinization. Results indicate that a 15 min-lysis time can achieve higher mtDNA yields from etiolated seedlings. Moreover, it is found that the inflorescence of the cytoplasmic male sterile (CMS) line is unfit for the isolation of mtDNA. The mtDNA isolated using this method is intact and pure, and can be used for further molecular analysis. Subsequently, the genomic and transcriptional differences of atps and coxs genes on the mitochondria between the petaloid-type CMS line and its maintainer line have been identified. RFLP analysis revealed that out of the five atps and three coxs genes, except of atp4 and cox3, the others mtDNA protein coding genes exhibited polymorphisms, respectively. This results suggest that atps and coxs genes are located in a long mtDNA fragment, and the mtDNA evolves rapidly in structure between the CMS line and its maintainer line in tuber muster. Northern blot analysis showed that the expression level of these genes in flower bud is higher than that of leaf and flower, and that, alternative splicing have been found among the atp6, atp8 and cox3 genes, respectively. Our results modified a efficient protocol for isolating the mtDNA, and provided some novel molecular markers indicating the CMS trait in tuber mustard. The comparative analysis presented in this study allows a more comprehensive understanding of the molecular mechanism on CMS in Brassica crops.