Sequencing And Analysis Of The Mitochondrial Genome For Puccinia Striformis F.sp.tritici

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most devastating fungal diseases in wheat,which severely threatens the security of wheat production worldwide.Breakdown of rust resistance due to virulence changes and new races occurred in the pathogen population of wheat stripe rust.And the genomic variation is the essence of virulence variation of stripe rust.Therefore,it is important to reveal the genome genetic variation of the pathogen including mitochondrial genome.With the development of the third generation DNA sequencing technology and the low cost of high-throughput sequencing,more and more pathogenic fungus completed the whole genome sequencing,but few studies about the mitochondrial genome sequencing were reported until now.As a genetic system outside the nucleus,mitochondrial DNA has the characteristics of integrity,polymorphism,small and self-replicating,which has important influence on biological development.The mitochondrial DNA also has a faster evolution of 5 to10 times than nuclear genome.The genome sequencing of Puccinia striiformis f.sp.tritici has been completed in the former studies,but the genome sequencing of mitochondria in Pst is still unfinished.This paper used both of the second and the third generation DNA sequencing technology to sequence and analyze the completed mitochondrial genomic sequence of Pst,and we also analyzed the relative expression of the important mitochondrial genes of atp6 and nad2 by real-time quantitative PCR.The main results were as follows:(1)The total DNA and mitochondrial DNA of P.striiformis f.sp.tritici race CY32 were extracted using the modified DNA extraction methods.The mitochondrial genomic reads were filtrated from the Pacbio data of the total genome DNA by the bioinformatics method.The filtered data were assembled into mitochondrial genome using the software of Celera Assembler sequence assembly.The bases of mitochondrial genome were corrected and the holes filling were processed by using the Illumina sequencing data with the gapcloser software.Finally,a total length of 81,777 bp of the completed mitochondrial genome sequences of Pst was obtained with 35.72% GC-content.(2)The Gene Mark software and BLAST sequence alignment were used to do the protein-coding gene prediction and its function annotation.And transfer RNAs were annotated by t RNAscan-SE search server.15 kinds of protein-coding genes and 24 transfer RNAs were obtained in total.It could be concluded that the quality of the completed mitochondrial genome was well,and Puccinia striiformis had a near relationship with Puccinia graminis,Phakopsora meibomiae and Phakopsora pachyrhizi according to the analysis of basal component,comparative genome,collinearity and the constructed phylogenetic tree,which laid a foundation for the study of gene function and species evolution of Puccinia striiformis f.sp.tritici.(3)From the analysis of relative expression of atp6 and nad2 genes in infection process,we discovered that the trends of the two genes in gene expression were the same and the two genes were closely related to the growing development and energy metabolism of Puccinia striiformis.At the early stage of pathogen penetration,the expression quantity of Pst-atp6 peaked early in the wheat confers nearly immune resistance against Pst,which indicated that Pst-atp6 was probably associated to the pathogenicity of Puccinia striiformis.At the later stage of pathogen sporulation,the expression quantity of Pst-nad2 was decreased under the expression level of urediospore only in the wheat confers nearly immune resistance against Pst.,The result indicated that Pst-nad2 was probably closed related to the mechanism of pathogen-plant interactions.