西藏牦牛mtDNA 16S rRNA基因的克隆及其遗传多样性分析

本研究首次通过克隆、测序获得了11个西藏牦牛类群共111头个体的mtDNA 16S rRNA基因全序列,并分析了西藏牦牛的遗传多样性、分类关系、起源和分化,为进一步保护和合理利用西藏牦牛遗传资源以及探讨牦牛类群的划分提供分子依据。结果表明：西藏牦牛16S rRNA基因全序列长度为1571 bp或1570 bp（LWQ4）;G＋C平均含量37.8%,具有明显的碱基偏倚性;平均核苷酸多样性（Pi）为0.00291,平均单倍型多样性（Hd）为0.8501±0.0009,111个样本共发现48种单倍型并聚为2簇,表明西藏牦牛具有较高的遗传多样性并存在2个母系起源;Kimura双参数遗传距离范围为0.00098-0.00694,11个西藏牦牛类群划分为2大类：嘉黎牦牛、巴青牦牛、丁青牦牛、工布江达牦牛、帕里牦牛、斯布牦牛、康布牦牛、桑桑牦牛、江达牦牛、桑日牦牛为一类,类乌齐牦牛单独为一类。本研究发现类乌齐牦牛具有较丰富的遗传多样性,并且发现了一个序列特异个体LWQ4,需要对其进行更深入的研究。牛种间的聚类结果显示,西藏牦牛与美洲野牛的亲缘关系最近,与普通牛、水牛的亲缘关系相对较远,本研究支持将牦牛从牛属（Bos taurus）中分离出来,作为一个独立的牦牛属（Poephagus）的观点。

Cloning and Phylogenetic Analysis of mtDNA 16S rRNA from Tibetan Yaks

The proper classification of Tibetan yaks is important to protect their survival and enhance Tibet＇s ecological balance. The mitochondrial DNA 16S ribosomal RNA（mtDNA 16S rRNA） gene was chosen to use as the molecular marker for our genetic diversity analysis of 11 Tibetan yak varieties（referred to below as groups,comprising total of 111 samples）. The yaks＇ mtDNA 16S rRNA genes were cloned and then positive recombinant clones were sequenced and analyzed by using various bioinformatics softwares, Based on analyses of genetic diversity, classification, origins, and differentiation, we found that the length of the complete mtDNA 16S rRNA gene in the Tibetan yak ranged from 1 570 bp to 1 571 bp. The average G＋C and A＋T content were 37.8% and62.2% respectively, showing a certain degree of base preference. Among the yaks, the average nucleotide diversity was a value of 0.002 91, which indicated a relatively high degree of genetic diversity within Tibetan yaks.Forty-eight haplotypes were detected in the 111 Tibetan yak samples; the entire haplotype diversity was 0.850 1＋0.000 9. The forty-eight haplotypes were divided into two clusters by constructing a network relationship chart,which suggested that there might be two maternal lineages of Tibetan yak. A phylogenetic tree based on Kimu ra-2 parameter genetic distances（0.000 98-0.006 94） showed that the 11 Tibetan yak groups could be divided into two clades. One is included the Jia Li yak, the Ba Qing yak, the Ding Qing yak, the Gong Bu Jiang Da yak,the Pa Li yak, the Si Bu yak, the Kang Bu yak, the Sang Sang yak, the Jiang Da yak, and the Sang Ri yak; and the other is only included the Lei Wu Qi yak. These results further suggest that the Tibetan yak has a relatively high level of genetic diversity, although the LWQ4 sample and the Lei Wu Qi yak group should be researched further. In a subsequent cluster analysis of several bovine species based on the mtDNA 16S rRNA gene, the Tibetan yak showed a close phylogenetic relationship with Bison bison but