Sequencing And Analysis Of Ribosomal ITS And Mitochondrial Genomics Of Rhipicephalus Microplus

Cattle tick(Rhipicephalus microplus)is the most common tick of livestock,especially cattle.This tick not only causes dermatitis and blood loss by bite,but also was considered as a vector of many pathogenic microorganisms.Rhipicephalus microplus have many branches(A-E branches).Accurate identification and differentiation of R.microplus and other closely-related Rhipicephalus species is very different based on their morphological features,hosts or geographical origins.In this paper,the ribosome ITSr DNA sequence and mitochondrial gene sequence were sequenced and analyzed by molecular biology method,in order to find appropriate molecular markers to accurately identify R.microplus.At the same time,the genetic evolution and gene polymorphism of R.microplus were studied.In the first part of this study,the sequence of transcriptional interval region(ITS)of R.microplus was amplified.To investigate whether ITS region of R.microplus can be identified with marker genes for identification and differentiation.In this study,35 R.microplus from Hunan,Guizhou,Hainan and Henan provinces were obtained,The ITS(ITS-1,ITS-2)region sequence of R.microplus were 1635 bp and 1151 bp in length,respectively.Based on the sequences of ITS(ITS-1,ITS-2)regions,the results showed that the intra-specific and inter-specific differences of the ITS(ITS-1,ITS-2)regions within R.microplus were 0-0.9%,0-0.3%,43.7-54.9% and 35.6%-40.7%,respectively.The results of our study suggested that the ITS r DNA as a good gene marker for tick identification and classification because the ITS r DNA sequence is more conservative and the difference of inter-specific among different species.However,it could not be used as a molecular identification of R.microplus complex species.The second part of the paper was analyzed the gene variation of three genes(cox1,nad1,nad4)of mt genome of R.microplus.From the perspective of mitochondrial genes,the genetic variation and DNA polymorphism of the mitochondria of R.microplus were studied.In order to find more specific genetic marker genes,which can be used to study the identification of species complex within R.microplus,and to explore the situation of species complex within R.microplus in China.The cox1,nad1,nad4 region sequence of R.microplus were 817 bp,350 bp and794 bp,in length,respectively.The results showed that the intra-specific and inter-specific differences of R.microplus were 0-8.3%(for cox1),0-4.3%(for nad1),0-10.3%(for nad4),12.2-17.1%(for cox1),18.6-21.1%(for nad1),23.3-25.4%(for nad4),respectively.The phylogenetic of R.microplus was reconstructed using the Bayes,MP and MLmethods.In the all phylogenetic trees,all the R.microplus isolates grouped together,indicating that all studied isolates represent R.microplus Clade A and B.For isolates from Hainan provinceclustered together in one clade(Clade A).Isolates from others three provinces(Hunan,Henan,Guizhou)clustered together in another clade(Clade B)without reflecting geographical origin.The study identified two cryptic branches from cox1,nad1,nad4 of R.microplus in China.Our result was consistant with previous studies based on 16 S genes.This result confirmed that cox1,nad1,nad4 genes could be used for the classification and identification of related species.In the third part of the paper,the entire mt genome of R.microplus wasannotated and analyzed.The complete mitochondrial genome of Chinese R.microplus Clade B has been included in the Gen Bank,but the complete mitochondrial genome of Chinese R.microplus Clade A not been reported in the Gen Bank.In this study,the mitochondrial genome of R.microplus Clade A from Hainan province was amplified by long PCR,and the mitochondrial genome of the PCR product was cloned,transformed and sequenced to obtained.The complete mt genome sequence of R.microplus Clade A was15163 bp in length,respectively.Both mt genomes include 13protein-coding genes,22 transfer RNA genes,two ribosomal RNA genes.The 13protein-coding genes of R.microplus have ATT,ATG,ATA as a start codon,and have TAA,T as a termination codon.The sequences of 13 protein-coding genes of each tick was combined a long sing sequence.The phylogenetic was analyzedusing three different computational algorithms ML.All results showed that the R.microplus located on the same branch in the phylogenetic tree.The results further support the existence of species complex within R.microplus.The results of this study not only enrich the contents of the mitochondrial genome of ticks,but also have important significance in studying mitochondrial taxonomy,biological evolution and populationgenetic structure of ticks.