Sequencing And Sequences Preliminary Analysis Of Cotton Mitochondrial Genome

The plant mitochondrial genome displays complex features, including large and highly variable sizes, the incorporation of foreign DNA sequences, recombinationally active repeats, the presence multiple subgenomic molecules and gene expression dependent on RNA editing. At the same time, an high incidence of illegitimate recombination in the mitochondria genome results in forming chimerical genes, some of which have been revealed through plenty of studies to have associated with cytoplast male sterility factor. In this study, sequencing and analysis mitochondrial genome of2074B, which is a maintainer line for gossypium hirsutum L. CMS, and pima90-53of gossypium barbadense L. were preliminarily studied with technique of high-throughput sequencing and genomic library.Through screening bacterial artificial chromosome (BAC) clones of mitochondria genome from G. barbadense,10positive clones containing mitochondria DNA fragments were obtained, and may basically cover all the mitochondria genome based on overlapping fragment and inserting fragment size analysis and prediction. One of these BAC clones was sequenced, in which abounding gene marks, and after splicing a115kb sequence was obtained. Gene annotation analysis revealed that this clone contained15reported functional genes of the mitochondria genome. Eight of these genes were chosen to make up of a linked gene cluster used for study on phylogeny of20plant species, which was to disclose evolutionary relation between these species objectively. Meanwhile, organization characteristics of this sequence and collinearity of its gene arrangement were analyzed, and RNA editing was predicted preliminarily.Using454sequencing technology the mtDNA of2074B were sequenced, we obtained the amount of data about114M, with an average reads length was399bp. Homology analysised with plant mitochondrial genome for large contig of sequences assmebledd by NCBI databases,110contig sequences have high similarity with the mitochondrial genome above800bp and total size is541.478kb, about42%of the analysis sequences. Meanwhile, we analyzed all of the contig sequences more than500bp and the number of homologous contig increased to133, total length is561.522kb. In view of the variability in intergenic regions of plant mitochondrial genome, we initially identified the133contig sequences as the mtDNA sequence of2074B.According to the composition characteristics of plant mitochondrial and2074A assembly sequences, we design primers for contig between the initial regions and89sequencing fragments assembling102contig to13larger contig sequences, the original133contig reduced to43and the total size increased to581.332kb. Genome annotation acquired54mitochondrial genes, including35protein coding genes and19RNA coding genes, these genes or introns located in the18contig. Repeat sequence analysis showed that the largest contig43repeats only488bp and more less100bp, so the acquisition of large fragments and accuracy assembly the repetitive sequences could be important step to next step complete the finsh map.In order to futher assembling those sequences, we constructed a2074B Fosmid mitochondrial genome library, which including2016clones and average insert of about40kb. Use62markers compsed with contig end and gene markers and6mixed pools of fosmid library, we screened28positive clones. Analysis found three clones that have overlapping relationship between the clone and assembly sequence, and the total number of the clones can be reduced to35after these clone sequenced. Analysis of the overlapping relationship with the clone and contig by markers, we found some conflicting connection order aslo exist, these connections whether display multiple molecular structure of mitochondrial, and how to assemble these sequences is undoubtedly essential to complete mitochondrial genome.In short, we have made a progress for the composition features and functional genes through mtDNA sequencing and library screening, and this work contribute for the further completion of the mitochondrial genome of G. barbadense and G hirsutum.