Molecular characterization reveals chlorosis-corrected CMS (Brassica oxyrrhina) B. juncea cybrid has recombinant mitochondrial genome involving male sterility inducing orf108-atpA gene

Alloplasmic Brassica juncea lines with B. oxyrrhina cytoplasm show stable cytoplasmic male sterility (CMS) but display chlorosis due to incompatible interaction between the plastid and the nuclear genomes. Normal, green CMS cybrids were developed through somatic hybridization between chlorotic CMS line and B. juncea. This study was aimed at characterization of the mitochondrial genome constitution of two chlorosis-corrected B. juncea cybrids. Comparative RFLP analysis of parents and cybrids with mitochondrial gene specific probes revealed B. oxyrrhina specific pattern in cybrids for atp4, ccmfn2, nad4L and cox3 but atpl and orf108 gave novel pattern indicating recombinant mitochondria. RT-PCR showed co-transcription of orf108 and atpl in the cybrids. PCR analysis with mitochondrial genome specific primers revealed that the cybrids had mostly B. oxyrrhina like mitochondrial genome. The two cybrids showed identical RFLP and PCR pattern suggesting that they are derived from the same event. Interestingly, RFLP pattern indicated two copies of orf108-atpl in the cybrid. One of the copies was derived from intermolecular recombination between B. juncea and B. oxyrrhina whereas the second copy resulted from intramolecular recombination between a pair of short repeat regions. The results show that orf108-atpl region is recombinationally active and could be involved in causing male sterility.