| Maize cytoplasmic male sterility,an important tool for heterosis utilization,always used as ideal materials for nuclear-mass interaction studies.Thus,Maize cytoplasmic male sterility has attracted serious concern of geneticists and breeders.C type cytoplasm male sterile of maize has anti maydis Race T and has stable fertility,whereas,lacking of a strong restorer line and the unclear mechanism of abortion and fertility restoring makes it difficult for large area production.Therefore,it is of great theoretical and practical significance to study the mechanism of abortion and fertility recovery of C-type cytoplasm male sterility of maize.In 2007,Allen et al.,had analyzed the effects of various cytoplasmic mitochondrial DNA on genome size,repeat,genes,ORFs and so on of five cytoplasmic mitochondria of NB,NA,CMS-S,CMS-T and CMS-C of maize via DNA sequencing.Massimo et al.(2005)also completed sequencing of the genotypes of the sterile and fertile cytoplasmic chloroplast genotypes of the above maize materials,and completed the T chromosome plastid genotyping.However,most of the previous studies analyzed the mitochondrial genome differences between a set of heteronuclear maize materials,which neglected that the cytoplasmic genome is mutated by the interactions of nuclear DNA and cytoplasmic DNA.In our study,two sets of homonuclear heterogeneous maize materials(C48-2,48-2,CHZS,HZS)and four homogeneous heteronuclear maize materials(C48-2,CHZS,C698-3,C478)were researched.The mitochondrial DNA of maize was extracted by modified centrifugation and density gradient centrifugation.The mitochondrial genome was sequenced by high throughput sequencing and the complete mitochondrial genome sequence was obtained by De novo assembly and Gap pooling.Then,mining genes that are difference in mitochondrial genome of two sets of homonuclear heterogeneous maize materials as the candidate gene that may cause cytoplasmic male sterility.Our study seek the cytoplasmic genome variation caused by nuclear and cytoplasmic interaction through the analysis of the cytoplasmic genome of heterogeneous heteronuclear material.key research findings are as belows:1.Mitochondrial genome sequencing data of C48-2,CHZS,C698-3,C478 and 48-2,HZS were more than 1GB after filtration,sequencing depth greater than 2000,which are in line with the assembly conditions.The results of genomic assembling were 739,660bp,739,653bp,739,607bp,739,643bp and 566,154bp,566,503bp,respectively,and the GC content was all about 44%.The results of chloroplast genome of C48-2,CHZS,48-2 and HZS were 140,473bp,140,478bp,140,458bp,140,448bp and GC content of 38.4%respectively.2.Compared with the sequence homology and collinearity of the mitochondrial genome of different materials,We found that the conserved region of the genotype C and the fertile material were higher in the conserved region of the mitochondrial genome,but the order and direction of these conserved sequence segments were different.There are large repeat sequences in the mitochondrial genome of C-type sterile materials,whereas the large repeat sequences in the fertile material are less than 241 kb.The results of the simple repeat sequence(SSR)also show that a large number of Five,six base units of the repeat sequence are in mitochondrial genomes,and the number in sterile materials are more than that in fertile materials.3.The mitochondrial genome of the six materials were annotated to 33 known protein-coding genes,The number of copies of nad4L,cob,cox3,atp9,rps4 genes in different cytoplasmic mitochondrial genomes was different through the blast of the the sequence of these genes.The base information of Atp4,atp6,Cox2,nad2,rps2B genes in different cytoplasmic mitochondrial genome were also different.It was found that the variation of the base sequence of the gene resulted in a change in the secondary structure of the protein and then change its function by analysing the predicted secondary structure and transmembrane domain of these differential gene.4.Two sets of heterogeneous maize chloroplast genes were all annotated to 84 protein coding genes,30 tRNA genes and 4 rRNA gene except that there were differences in the copy number of some genes between different materials.Collinearity comparative analysis found that the structure of chloroplast genome was highly conserved,and little sequencing of sequence fragments was recombined.Analysis of the variation sites(SNP and InDel)on the chloroplast genome of the four materials showed most single base variation and all insertion and deletion occurred in the genomic Poly structure region.5.Comparing the structural information,repeats,coding genes and mutations of mitochondria and chloroplast genomes of two sets of homonuclear heterogeneous maize materials(C48-2,48-2,CHZS,HZS)illuminated that the mitochondrial genome in sterility were more complex than in the fertile.The main reason may be that it has a large number of repetitive sequences,while the chloroplast genome is highly conserved.Comparing the mitochondrial and chloroplast genomes of maize showed that different nuclear background had an effect on mitochondria and chloroplast genomes in the same cytoplasm,but these differences mainly occurred in the repeat sequence and the gene septal region and only few cases will affect the copy number of the gene.Meanwhile,the gene base sequence and its encoded product structure has not been affected. |
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