Assembly Of The Mitochondrial Genome Of Ophiocordyceps Sinensis And Its Comparative Genomic Analysis

Chinese cordyceps,one of the most important valuable Traditional Chinese Medicine,is composed by an fungus(Ophiocordyceps sinensis)parasitizing the ghost moth Thitarodes larvae,which live in alpine meadows of the Tibetan Plateau.O.sinensis is an ideal material for fungal evolution research,due to the different climate types and terrain among different regions on the Tibetan Plateau,and abundant species diversity of host insects.Mitochondrial DNA is a powerful tool to study the origin and evolution of organisms.Because of the conservation of genetic structure and composition,low mutation rate and fast evolution,mitochondrial DNA is an ideal molecular marker for population genetics and molecular evolution.To date,mitochondrial genomes(mitogenomes)of more than 180 fungal species have been released in the NCBI,but there is no data of O.sinensis.In this paper,we assembled the complete mitogenome of O.sinensis strain XZ08-51-18 by PCR according to the published reference genome sequencing data of O.sinensis(CO18 strains).The mitogenome of entomopathogenic fungus O.sinensis,with a total size of 157559 bp,is one of the largest mitogenome among sequenced fungi,and is only smaller than that of Rhizoctonia solani(235,849 bp in basidiomycetes)and Sclerotinia borealis(203,051 bp in ascomycetes).As with most of the fungal mitochondrial genome,it contained the 14 typical genes coding for proteins,containing three subunits of cytochrome C oxidase(cox1,cox2,cox3),three subunits of ATP synthase(atp6,atp8,atp9),cytochrome-b gene(cob),seven subunits of NADH dehydrogenase complex(nad1,nad2,nad3,nad4,nad4L,nad5,nad6),two subunits of rRNA(rnl and rns),and 27 tRNA genes,as well as two rRNA genes.The entire mitogenome contains 54 introns,the ORF which code some Homing Endonucleases(GIY-YIG and LAGLIDADG),four reverse transcriptase,one ribosomal proteins and three unknown enzyme.We found RNA polymerase and transposase in intergenic region,and introns and intergenic region accounted for about 86%of the entire genome.Compared with all reported mtDNAs of hypocreales,it illustrated a highly conserved genome organization for all the protein-coding genes and rRNA genes,as well as three clusters of tRNA genes between rnl and nad2,rns and cox3,nad6 and rnl.A phylogenetic study of O.sinensis in hypocreales strongly supported a view that traditional Cordyceps fung were divided into different genera.In order to analyze the mitochondrial differentiation of O.sinensis,we choose eight strains of O.sinensis from different geographical locations for genome re-sequencing,and then design primer by referring to the XZ08-51-18 mitochondrial genome sequence,PCR amplification,and sequencing.Now we got eight O.sinensis complete mitochondrial genomes.Then,gene annotation and different O.sinensis strains of mitogenome organization comparison,arrangement,and nucleotide mutation gene were carried out.The results show that all O.sinensis contained the 14 typical genes coding for proteins,25-27 tRNA genes,as well as two rRNA genes.The PCGs are arranged in sequence according to a certain order of rnl-nad2-atp9-cox2-nad4L-nad5-cob-cox1-nad1-nad4-atp8-atp6-rns-cox3-na d6.Nad2 and nad3 are directly connected and there is one overlap of the base A between nad4L and nad5.To compare nine O.sinensis mitogenome sequences,we found mitogenome were similar,but the lengths were different.The primary reason is that intron insertions in certain genes and different length of sequences insertion of the intergenic regions.Large differences loci were verified by Electrophoresis to screen more efficient molecular marker genes(rnl-i5,rnl-E2,cox1-i5,cob-E6,rns-E1,nad2-i3,cox2-i5,rnl-E5,rnl-i6).14 conservative sequences of exon of protein-coding genes were used for phylogenetic analysis.The results showed that different strains of clustering relation had certain correlation with the evolutionary branch among analysis of multiple genes of O.sinensis,but it didn't match exactly.The aims of study were to report mitogenome organization of O.sinensis providing the reference sequence of mitogenome for this specie.Meanwhile,we compared with mitogenome sequence of nine strains of O.sinensis and other fungi for improving understanding in evolutionary rules of fungal mitochondrial DNA.It is important to study the evolution of O.sinensis that contribute to our understanding of the origin and spread of O.sinensis,as well as developing protection measures of wild O.sinensis.