Mitochondrial Genome Sequencing And Analysis Of Cytoplasmic Male Sterile Line And Its Maintainer Line In Soybean

The seeds of soybean, rich in nutrients, are not only very useful for health, but also the world's most important grain and oil crops. Faced with the situation that the soybean production lags behind, supply shortages and import increases year by year, to improve soybean production is critical. Cytoplasmic male sterility (CMS) is a negative trait to the plant, but is good for breeding. Therefore, carried out the research of molecular mechanism of cytoplasmic male sterility in soybean has important theoretical and practical significance for make better use of heterosis in soybean.A large number of research results show that the CMS has a direct relationship with mitochondrial DNA genome, however, recently, the studey of mechanism of CMS-related research is not enough. Frequency of mitochondrial genome reorganization and code redundancy, genome structure and size vary greatly, make the research work more difficult. With the development of the molecular biology techniques and theory, we have found a number of genes associated with CMS, but because the CMS involves very complex factors, so the current study is not enough to explain the relevant mechanism. To better understand these mechanisms, we need a whole knowledge of these genes. The reports of mechanism of cytoplasmic male sterility in soybean-related are few, the research is lagging far behind, compared with corn, rice and other crops. So, the construction of mitochondrial genomic library and sequencing, will lay the foundation for research of soybean cytoplasmic male sterility mechanism, and will improve proteome research and comparative genomics.The purity and concentration of mitochondrial DNA would infect the result of Shotgun sequencing and the construction of BAC library, so, in this study, cytoplasmic male sterile line of soybean (JLCMS1-1A) and its maintainer line (JLCMS1-1B) were planted in dark, then we used these seedlings to purify mitochondria. The mtDNA was proved suitable for genome sequencing, high purity without nuclear DNA and Chloroplast DNA. In order to determine whether there were nuclear DNA and chloroplast DNA or not, we use 3 pairs of primers, designed by known genes of soybean NARK, psbC, and CO, respectively amplified the nucleus, chloroplast and mitochondria-related genes. Confirmed by PCR identification, the soybean mitochondrial DNA is High purity and suitable for genome sequencing to the next step. We constructed Shotgun libraries for the cytoplasmic male sterile line of soybean and its maintainer line, then extracted the plasmid in large-scale. Sequenced the genome 8-10 times by using fluorescently labeled dideoxy method, through MegaBACE 4500 sequencer, M13 universal primers Sequenced in both directions. The results spliced with the Phred/Phrap/Consed program through the computer group which is in parallel in the RedHat Linux platform. The complexity of the structure of mitochondria and their highly repetitive sequences made the assembly of the sequence difficult, so finally the whole genome only spliced into several large contigs (contig). Therefore, after Shotgun sequencing, primer walking method was used to fill gaps.We constructed a BAC library of the maitainer line, in order to establish the physical relationship of these contigs, and then fill the physical gap. The average insert size of library was 50Kb, preserved 2,000 clones, about 250 times of the entire mitochondrial genome of the maintainer line. Designed a primer at both ends of each to amplify the unknown sequence, roughly determined the position relationship between contigs by PCR reaction; in the end of each contig designed a pair of primers, looking for the BAC clones across "Physical channel" by PCR reaction. Referenced to the results of BAC end sequencing, finally determined the clone and contructed Shotgun library of it, then sequenced and spliced to fill the physical gap. After these work, we finally got 7 contigs of the mitochondrial genome of CMS line, total length about 375Kb, the longest continuous sequence 122,948bp; 6 contigs of the maintainer line, total length about 400Kb, the longest continuous sequence 214,737bp, which is by far the longest sequence of the mitochondrial genome of soybean.Bioinformatics analysis showed that the soybean mitochondrial genome sequence of G +C content of CMS line and its maitainers line was 45.12% and 44.93%, more than 85% of the two mitochondrial gene segments is consistent with high homology. Gene annotation and blast analysis showed that:they both coded 31 kinds of protein, including mitochond-rial cytochrome oxidase, NADH dehydrogenase, ATP enzymes,3 rRNA and 13 tRNA. The gene nadl, nad2 and nad5 contain multiple exons, across large genomic regions just the same as many mitochondrial genome in other species. In this study, firstly, we sequenced CMS line and its maitainer line of soybean and analysed the gene. The comparison of the two mitochondrial genome and gene annotation, will prove many data for looking for Cytoplasmic Male Sterility related genes. We also constructed a BAC library of the maintainer line mitochondrial genome, it is not only useful for post-specific gene map-based cloning and cytoplasmic research, but alse good for the research of soybean mitochondrial functional genomics, proteomics and comparative genomics. But in the end, the sequence of the two mitochondrial genome still exist "ditch", filling these gaps is necessary for further analysis for the related mechanism of cytoplasmic male sterility in soybean. When get the complete mitochondrial genome sequences of soybean male sterile line and its maintainer line, it can not only provide the firse-hand information on gene sequences for the molecular mechanism, but also promote the gene cloning, gene structure and function studies in the future.