The Mitochondrial Genomes And Functions Of Hau Cytoplasm Male Sterility Gene In Brassica

Cytoplasmic male sterility(CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. Therefore, breeders and biologists have long been interested in the genetic and molecular mechanisms of CMS. And pol CMS and ogu CMS are the main CMS systems used for hybrid seed production in B. napus. Meanwhile, there are lots of different CMS mitotypes found in Brassica, including: nap, Tour CMS, Moricandia arvensis CMS, Nsa CMS, NCa CMS, hau CMS, inap CMS cytoplasms et al.Cytoplasmic male sterility is mainly caused by the chimeric genes located in mitochondrial genomes. But the molecular mechanism of CMS in B. napus is still elusive. The hau CMS line has a complete and stable male sterility, and it also has great potentials in hybrid seeds production. Based on the hau CMS line, its iso-nuclear maintainer line and the restore of F1 hybrid plants in B. napus and B. juncea, in this research, we sequenced and comparatively analysis the mitochondrial genomes of hau CMS line and the other mitochondrial genomes in Brassica. And primary analysis the molecular mechanism of the hau CMS line through cytology, genetics and genomics. The main results are as follows:1. Based on the cytological study, the abnormal anther development in the hau CMS line is arrested at the differentiation of the archesporial cells. And the stamen abortive thoroughly and no pollen sac appeared. Numerous defective mitochondria appeared in male sterility anther with cells swollen and the cells of sterile anthers contained more vacuoles. And the cell death eventually and caused totally male sterility.2. The mitochondrial genomes of hau CMS line and its iso-nuclear maintainer line had been sequenced. The hau CMS mitochondrial genome was highly rearranged when compared with the mitochondrial genome of its iso-nuclear maintainer line. Comparative analysis of it with the other sequenced mitochondrial genomes in plants showed that the mitotype of hau CMS is a heterologous cytoplasm.3. Different mitotype specific ORFs had been found after comparative analysis of mitochondrial genomes between the hau CMS line and its iso-nuclear maintainer line in B. juncea. Mitotype specific ORFs were used to develop specific markers to separate them at the seedling stage. It was confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its iso-nuclear maintainer lines in B. juncea. After comparatively analyzed the sequenced mitochondrial genomes in seed plants, we found that the mitochondrial genomes of B. nigra and B. carinata divergence from the mitochondrial genomes of B. rapa, B. oleracea, B. napus and B. juncea. And it may confirmed the lineage divergence of the B genome chromosomes could be distinguished from chromosomes of the A and C genomes in Brassica.4. After comparatively analyzed 6 sequenced mitochondrial genomes in Brassica napus, 90 mitotype-specific sequences(MSSs) of these mitochondrial genomes had been found. Additionally, 12 mitotype-specific markers were developed based on the mitochondrial genome specific sequences in order to distinguish among these different mitotypes. And use of molecular markers to identify different mitotypes in Brassica napus could enhance breeding efficiency. Cytoplasms of 570 different inbred lines collected across scientific research institutes in China were identified using these MSS markers developed in our study. The cytoplasm types of 533 inbred lines were authenticated. And the most prevealent cytoplasms in B. napus were the nap and pol CMS mitotypes. And there may be unidentified mitotypes existed among the 37 inbred lines.5. Northern blot hybridization and q PCR results showed that the expression pattern of orf288 in male sterility line and its fertility restore line differ apparently. But the expression pattern of the other two mitotype specific ORFs, including orf325 and orf220 had not been changed at transcription level. The CMS associated gene orf288 inhibit the growth of E.coil(toxalbumin). The toxicity region mainly concentrated in the three transmembrane domains of orf288. Genetically modified experiment with the constitutive vector transformed in B. juncea and Arabidopsis further confirmed that orf288 is the key gene which caused male sterility. And the core region which caused male sterility is mainly located in the C-terminal of the protein, but not in the N-terminal. This result may suggested that the cytoplasmic male sterility in hau CMS is not associated with toxic effect. orf325 had no relationship with the male sterility of hau CMS line; And the relationship between orf220 and the male sterility of hau CMS line need to be further studied.6. After comparative transcript profiling of flower buds from hau CMS line and its iso-nuclear maintainer line, some unigenes belong to the MADS box family controlling early anther development were dramatically down-regulated in hau CMS buds. Genes involved in reduction-oxidation reactions, cell metabolic and protein synthesis pathways were down-regulated.