Analysis of mitochondrial recombination in the male sterile Brassica juncea cybrid Og1 and identification of the molecular basis of fertility reversion.

KEY MESSAGE: Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility. An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.