The Analysis On Complete Mitochondrial Genome Sequence Of Rhipicephalus Turanicus And Construction Of Novel Genetic Marker For Intraspecies Identification Of R.turanicus

Object:(1)This study is aimed to determine mitochondrial genome sequence of Rhipicephalus turanicus,analyze its structural feature and the phylogenetic relationship among Rhipicephalus ticks.(2)Three novel mitochondrial genetic markers for R.turanicus intraspecific molecular identification were designed.Methods:(1)Primers of six mitochondrial fragments based on mitochondrial genome sequences of Rhipicephalus from the Gen Bank were designed to PCR amplify mitochondrial genome sequence of R.turanicus from China.Many softwares were employed for analysis of complete mitochondrial genome sequences of R.turanicus and phylogenetic relationship of ML method among hard ticks was analyzed.(2)Two hundred and twelve adult R.turanicus ticks infesting sheep from seven counties and one hundred and three adults from four counties,Albania were collected.All of the ticks were firstly underwent by morphological method and molecular analysis based on partial mitochondrial(16S r RNA and cox1)gene sequences.Phylogenetic analysis of ML method was then conducted.Primers of three mitochondrial genetic markers,namely partial nad1-16 S r RNA(N1),nad2-cox1(N2),cox1-t RNA-Lys(C1)mitochondrial fragments based on conserved sequences of complete mitochondrial genome of Rhipicephalus ticks were designed for further study.Altogether 315 ticks of R.turanicus were included.Three fragments(N1,N2 and C1)were PCR amplified for intraspecific genetic divergence and phylogenetic relationship analysis in this study.Results:(1)For the first time in the world,mitochondrial genome sequence of R.turanicus,which is 14717 bp in size and contains 13 protein-coding genes,two non-coding regions,22 transfer RNA genes,a small subunit ribosomal RNA gene(rrn S)and a large subunit ribosomal RNA gene was sequenced successfully.The mitochondrial gene arrangement of R.turanicus is consist with that of Metastriata ticks.The complete mitochondrial nucleotide is composed of A(37.80%),T(40.00%),G(9.90%)and C(12.30%),and A+T contents is 77.80%.ML phylogenetic tree showed that R.turanicus located at Metastriate branches and was closest with R.sanguineus from China on genetic relationship.R.turanicus and R.sanguineus from China were sister with R.sanguineus from USA on genetic relationship.(2)Three newly genetic markers(N1,N2,C1)is a valid genetic tool on intraspecific genetic divergence of R.turanicus.The divergences from Chinese-Albania interface was 3.57-4.07%,3.57-4.39% and 3.18-4.69%,respectively.Two different subspecies of R.turanicus were divided by Three newly genetic markers(N1,N2,C1)in this study.Conclusion:(1)Around the world,mitochondrial genome sequence of R.turanicus was firstly sequenced which is circular-close,14717 bp in size and contains 13 protein-coding genes,two non-coding regions,22 transfer RNA genes,a small subunit ribosomal RNA gene(rrn S)and a large subunit ribosomal RNA gene and one copy.Presently,ML phylogenetic tree showed that R.turanicus was closest with R.sanguineus from China on genetic relationship.R.turanicus.More mt DNA in Rhipicephalus should be completed in further study.(2)Three newly genetic markers,in this study,(N1,N2,C1)is a valid genetic tool on intraspecific genetic divergence of R.turanicus.Herein,at least two different R.turanicus subspecies might exist in the world.In the future,more efforts are needed to prove this,based on a larger number of R.turanicus specimens collected throughout its vast geographical range.