Sequencing Mitochondrial Genomes And Preliminary Analysis On Mechanism Of Cytoplasmic Male Sterility In Cotton

Cotton is one of the most economical crops.Cotton has a very significant heterosis that can improve the yield and quality of cotton.The current studies show that cytoplasmic male sterility(CMS)is closely related with mitochondrial genomes(mitogenomes).Generally,comparing the genome,transcriptome and proteome between sterile lines,maintainer lines and F1(contain restoring genes)to looking for the CMS genes.Obtained mitogenome sequence can be directly analysis the mechanism of sterility from primary structure;in addition,the sequence of mitogenomes would provide new information to explore the phylogeny and relationships of these cotton genuses and provide a theoretical basis for the interaction of cytoplast and nuclear in hybrid breeding.This study contains extraction of mitochondrial DNA,construction of genome library,genome sequencing and transgenosis,and had preliminary genetic analysis on the cytoplasmic male sterility of G.harknessii and upland cotton.The main achievements and results of my study were as follows:1.We used etiolation seedling and callus for extraction the cotton mitochondrial,and used Mito-track to dye the mitochondria,which were observed intact and little pollution with fluorescence microscope;then we obtained the mtDNA and mtRNA,which meat the requirements of further experiments.2.Construction of four fosmid libraries of mitogenomes;the libraries consisted of 1152-2016 clones with an average length of insert fragments of 38 kb,covering 61-108 fold of mitogenomes.E3.We had screened the libraries by 28 pairs of primers from functional genes and scaffolds,and constructed the physical maps of mitogenomes.4.Using high-throughput sequencing technologies and physical maps,we obtained four complete mitogenomes sequences,whose size was from 621.884 to 668.584 kb,GC content is 45%,and contains 37 protein-coding genes,30 tRNAs and four rRNAs.These genomes have 4-7 larger repeats,which were almost covered by positive clones and no subcycles produced by them.Based on the evolution analysis of mitogenome and chloroplast genome found that the 2074 S,E5903 and 2074 A were closer;2074B was relative far from these three variaties.5.We forecasted ORFs(open reading frame)(>300 bp)in four genomes,and found 33 unique ORFs in two sterile lines;according to the characteristics of CMS(cytoplasmic male sterility)genes,we analyzed all of the ORFs in detail and finally obtained four candidate ORFs for cotton sterility genes.6.A specific orf160 in downstream of atp4.The subcellular localization of onion epidermal cells showed the green fluorescent were on cell membranes,so the mitochondria-targeting carrier can normal expression.The expression vectors were constructed and transformed into yeast,arabidopsis,tobacco and cotton,respectively.The plaque of modified yeast was abnormal and grew slower compared with control;the most seeds of transgenic plants of arabidopsis and tobacco were abnormal and shriveled;and the seed setting rate,germinative force and pollen grain vitality were lower compared with wild control.There was no sterile plant in transgenic cotton,but the plant height was lower.All these results showed that orf160 had an effected on the development of plant,and further research need to verify the relationship of orf160 and cytoplasmic male sterility in cotton.7.We constructed an interference vetor of atp4,and transformed it into sterile lines 2074 A and 2074 S,and restorer lines E5903,half of the positive plants was fertility in transgenic progeny of 2074A;and 2 sterile plants were found in the transgenic offspring of restorer lines E5903,so we speculated that atp4 may related to cytoplasmic male sterility of G.harknessii.