中国新疆吐鲁番地区小亚璃眼蜱遗传进化及单倍型分析

Genetic evolution and haplotype analysis of Hyalomma anatolicum in Turpan region of Xinjiang,China

为探究中国新疆吐鲁番艾丁湖镇(后简称吐鲁番地区)小亚璃眼蜱的遗传进化特征及单倍型,本研究提取10份来自中国吐鲁番地区已经形态学鉴定为小亚璃眼蜱的DNA作为模板,采用本研究室设计的中国新疆地区优势璃眼蜱种16S r RNA基因通用引物,通过PCR进一步鉴定上述小亚璃眼蜱并测序;采用DNAMAN V 5.0软件分析吐鲁番地区10份小亚璃眼蜱16S r RNA基因与来自中国新疆(阿瓦提)、巴基斯坦、印度小亚璃眼蜱相应基因的同源性;经MEGA X软件构建小亚璃眼蜱与上述地区来源的小亚璃眼蜱16S r RNA基因的系统发育树,分析小亚璃眼蜱种的亲缘性和遗传多样性。PCR结果显示,10份小亚璃眼蜱均扩增出388 bp的目的基因片段,经BLAST比对结果显示10份样品均为小亚璃眼蜱;16S r RNA基因序列的同源性分析结果显示,本研究采集的10份小亚璃眼蜱之间的同源性最高,达99.57%,与来自巴基斯坦、印度、中国新疆(阿瓦提株MG651939)小亚璃眼蜱的同源性分别为99.55%、99.17%及99.11%;16S r RNA基因的遗传进化分析结果显示,中国新疆吐鲁番的小亚璃眼蜱1、3、9及10聚为一小支,遗传距离较近;小亚璃眼蜱2、4~8与巴基斯坦、印度及中国新疆小亚璃眼蜱聚为一大支,遗传距离较近。提示,中国吐鲁番地区的小亚璃眼蜱可能来自巴基斯坦。采用DNA SP V5.0软件统计吐鲁番及上述国家或地区小亚璃眼蜱的单倍型数量(Np)、单倍型多样性(Hd)及核甘酸多样性(pi);采用Popart v1.7软件构建吐鲁番及上述国家或地区小亚璃眼蜱的单倍型网络图。结果显示,来自4个不同国家或地区小亚璃眼蜱的Np为8,分别为Hap_1~Hap_8,Hd为0.572、pi为0.01156。与其他3个地区相比,中国吐鲁番地区小亚璃眼蜱的Np最高为7、Hd为0.911、pi为0.02593。小亚璃眼蜱单倍型网络图显示,在4个国家或地区小亚璃眼蜱仅共享单倍型Hap_1,其他3个地区存在单倍型Hap_2~Hap_8。中国吐鲁番地区小亚璃眼蜱不仅共享单倍型Hap_1,还存在单倍型Hap_3~Hap_8。上述结果表明吐鲁番地区小亚璃眼蜱可能来源于中国新疆阿瓦提地区及巴基斯坦,且其遗传多样性较为丰富。本研究为了解蜱种群间的种属鉴定和遗传进化关系等提供参考依据。

In order to explore the genetic evolutionary characteristics and haplotypes of Hyalomma anatolicum in Aiding Lake Town of Turpan(abbreviated as Turpan region)in Xinjiang,China,this study extracted 10 individual DNA samples from Turpan of China that have been morphologically identified as H.anatolicum.The universal primers for the 16S rRNA gene of the dominant Hyalomma species in Xinjiang of China designed by our laboratory were used to further identify the above H.anatolicum by PCR and followed by sequencing.DNAMAN V 5.0 software was used to analyze the homology of 16S rRNA genes of the 10 H.anatolicum from Turpan with those from Xinjiang(Awati)of China,Pakistan,and India.The phylogenetic tree of the 16S rRNA genes of H.anatolicum and those from the aforementioned was constructed by MEGA X software to analyze the phylogenetic and genetic diversity of the tick species.The PCR results showed that 388bp target gene fragments were amplified from all 10 samples of H.anatolicum.The BLAST comparison results showed that all 10 samples were H.anatolicum.The homology analysis of the 16S rRNA gene sequences showed that the highest homology among the 10 samples collected in this study was 99.57%,and the homology with the samples from Pakistan,India,and Xinjiang(Awati strain MG651939)of China,was 99.55%,99.17%,and 99.11%,respectively.The genetic evolution analysis of the 16S rRNA gene showed that tick 1,3,9 and 10 from Turpan region,Xinjiang of China,were clustered into a small branch with a relatively close genetic distance,and tick 2,4,and 8 from Pakistan,India,and Xinjiang of China,were clustered into a large branch with a relatively close genetic distance.It is suggested that H.anatolicum from Turpan region of China in this study may come from Pakistan.DNA SP V5.0 software was used to count the haplotype number(Np),haplotype diversity(Hd)and nucleotide diversity(pi)of the tick in Turpan and the aforementioned countries and regions.Popart v1.7 software was used to construct a haplotype network diagram of the tick in Turpan and the aforementioned areas.The haplotype statistical results showed that the Np of the tick from four different countries and regions was 8,namely Hap_1-Hap_8,respectively,Hd was 0.572 and pi was 0.01156.Compared with the other three regions,the highest Np of the tick in Turpan region in China was 7,Hd was 0.911 and the pi was 0.02593.The haplotype network diagram of the tick from the four countries and regions showed that only Hap_1 was found in the four countries and regions,while the haplotypes Hap_2 to Hap_8 were found in the other three regions.The H.anatolicum in Turpan of China not only shares haplotypes Hap_1,but also has haplotypes Hap_3-Hap_8.The above results indicated that H.anatolicum in Turpan region of China may originate from Pakistan and its genetic diversity was relatively rich.This study provides a reference basis for understanding the species identification and genetic evolutionary relationship among tick populations.