Studies On Mitochondrial Genomes Of The Two CMS Lines In Brassica Napus L.

Cytoplasmic male sterility (CMS) is one of main tools for utilizing hybrid heterosis. And as a natural occurrence of sterility, CMS is also a good material for genetical research in higher plants, so it has attracted much attention from plant scientists. Lots of researches have demonstrated that CMS is caused by structural differences of the mitochondrial DNA between fertile and sterile cytotype. Pol sterile cytoplasm is most widely adopted in wordwide canola hybrid-seed production. Present studies focused on mitochondrial genome sequencings for two different cytoplasms that were Pol cytoplasm and newly developed sterile cytoplasm in Brassicca napus. Further, through comparisons among three subgenomic sequences, the structural genome differences were researched. Constitutes in mitochondrial genomes of rapeseed have been detected. Results are as following:1. Mitochondrial genome of a Pol CMS line NH12A.①Genome sequencing and the gene identification. NH12A developed by our lab is a Pol CMS line in B. napus L. Using shotgun sequence strategy, we sequenced its entire mitochondrial genome. A total of 2592 reads were obtained through randomly sequencing the clones, covering about 11 times of the mitochondrial genome size. The numerous sequences were further assembled into five contigs by Phred-Phrap software and the gaps were further closed by PCR. The completed circle mitochondrial genome is 223,412 bp in size. In order to distinguish the mitochondrial genome from Nap cytoplasm, we named our sequence pol mitotype and renamed the mtDNA sequence previous reported as nap mitotype.34 protein-coding genes,3 ribosomal RNA genes and 18 tRNA genes were identified in pol mitotype. The total genic length accounts for 17.34% of pol mitotype sequence.48 out of the 55 genes in pol mitotype are identical to that of nap. Pol mitotype have two copies of trnH gene, but nap mitotype has only one copy. Except for orf224, the other 6 genes slightly different from its counterpart genes in nap mitotype were cox1, cox2, atpl, trnC and trnE genes with no influence on gene function.44 putative ORFs longer than 100 codons were also detected in pol mitotype. Results from comparing the 44 ORFs with annotated ORFs of nap mitotype, showed that 35 ORFs are identical, but the other ORFs are different. The orf122 and orf132 are only present in pol mitotype and orf117b is unique in nap mitotype. ②Comparative mitochondrial genome analysis between pol and nap mitotype. Alignment of pol and nap mitotypes showed that the sequences have high identities, but their syntenic region orders are distinctly different. At least 5 irreversible rearrangement events were postulated to account for the structural differences, and CMS-related orf224 neighboring region about 5 kb rearranged twice with pol mitotype sequence as reference. Additionally,3.63% of nap mitotype and 4.53% of the pol mitotype cannot be found in their counterpart mitotype.③Analysis of repeated sequences and SNPs. In pol mitoytpe, lots of repeats were discovered. The repeats include a pair of larger repeats with 2427bp, short repeats accounting for 4.96% of whole mitochondrial genome, and 19 groups of small tandem repeats. The pair of larger repeat is related to formation of mitochondrial genome multipartite structure formulated through reversible recombination. The short repeats are closely related to invertible rearrangement of mitochondrial genome leading to novel ORFs. SNPs analysis between pol and nap mitotypes showed that there are 197 SNPs counting as a polymorphism rate of 8.88 bp in 10,000 bases. But except orf224/orf222 region, there are only 6 SNPs in the function-known genes, indicating that the mitochondrial genomes are highly conservative and stable in function-known gene sequences.2. Detection of mitochondrial genome constitutes①Coexistence determination of pol and nap mitotypes. We designed 11 pairs of mitotype-specific PCR primers according the two entire mitotype sequences to finish experiments with 16 samples of mtDNA from different genetic materials including 6 Pol CMS lines, their maintainers and 4 canola varieties. PCR amplification showed that all primers can amplify target fragments no matter that it was from the CMS line or maintainer. Hence, the experiments consistently demonstrated that both pol and nap mitotypes coexist in B. napus.②Quantitative detection of copy numbers of orf222 and orf224. TaqMan qPCR assays revealed that in Pol CMS lines, the contents of orf224 were more than orf222, but orf222 content always preponderated over that of orf224 in their maintainers and other fertile varieties. The results indicated that in nap cytoplasm, nap mitotype was as the majority genome companied with the pol mitotype as sublimons, and vice versa in Pol cytoplasm. Interestingly, there was two to six orders of magnitude variation of the copy-number ratio under the same cytoplasm. The dramatic variations of substoichiometric ratios for accessions with the same cytoplasm may probably be subscribed mainly to nuclear genotypes. The difference substoichiometric ratios between different years indicated that environmental factors may also play roles in deciding the copy-number ratio. Additionally, there is substoichiometric shifting of pol and nap mitotype in mitochondria of rapeseed which may cause the male fertility shifting. The coexistence of pol and nap mitotype in mitochondria and substoichiometric shifting provide a solution to the decades-existing and perplexing issue on CMS discoveries by different plant breeders.3. Mitochondrial genome of a new CMS line N219A.①Mitochondrial genome sequencing and the gene identificationBy solexa sequencing technology, we obtained mitochondrial genome of a new cytoplasm line N219A developed from somatic hybrids between B. napus and Descurainia sophia. N219A mitochondrial main genome was a single circle DNA molecule in size of 261671 bp which contained 33 protein-coding genes,3 ribosomal RNA genes and 17 tRNA genes. Comparing with nap mitoytpe, N219A mitochondrial main genome missed orfB gene and had a trnY gene which is not present in nap mitotype.49 putative ORFs longer than 100 codons were also detected in N219A mitochondrial genome. Results from comparing the 44 ORFs with annotated ORFs of nap mitotype, showed that 31 ORFs of them are identical, but the other ORFs are different. orf153, orf195b, or/130, orflOlf, orf122 and orf127 are unique in N219A mitochondrial main genome.②Comparative mitochondrial genome analysis between nap mitotype and N219A. Indel sequences are frequent between the two genomes. N219A mitotype lose 4.7kb sequence of nap mitotype, but gained 39.6kb sequence with unknown-original of which 55.96% sequence of insert sequence has homologous with other plant mitochondrial genome. N219A mitotype contained more repeated sequences which accounting for 10.6% of whole mitochondrial genome than nap mitotype. The differences between N219A mitochondrial genome and nap mitotype may probably be resulted from recombination in somatic hybrids. Identification of SNPs between N219A and nap mitotype showed that there are 305 SNPs showing a polymorphism rate of 13.7 bp in 10,000 basesSummarily, the comparisons of mitochondrial genomes confirmed the differences of cytoplasm mitochondrial genomes, and our experiments revealed the popularly coexisting of two mitochondrial sub-genomes within rapeseed. The conclusions have provided new theoretical suggestions on CMS in B. napus.