基于SANDX-2序列变异和SLA-DQB第2外显子的PCR-SSCP分析中国大陆地区野猪种群遗传多样性

【作者】 霍爱玲；

【导师】 常青；

【作者基本信息】 南京师范大学 ， 动物学， 2006， 硕士

【摘要】 野猪(Sus Scrofa)是家猪的祖先，主要分布于亚洲、欧洲及非洲的大部分地区。本文首先对单拷贝核DNA标记(single copy nuclear DNA，scnDNA)在种群遗传学研究中的应用进行综述，然后分别基于SANDX-2序列变异和SLA-DQB第2外显子的PCR-SSCP带型，初步分析中国大陆地区野猪种群的遗传多样性，并对影响SLA-DQB进行SSCP分型的实验因素进行了优化。具体内容如下： 1、研究测定了89个中国野猪和1个家猪未知功能核基因SANDX-2位点的序列。序列比对后长510bp左右，发现有2处4种类型碱基缺失，30个位点发生碱基替换，共定义了19种单元型。在野猪中共发现18种单元型，其中7种只在某一地方种群中发现，11种为地方种群间共享单元型。用邻接法基于核苷酸Kimura 2-parameter模型构建系统发生树中，19种单元型被明显分为2大支，但各枝得到的自展值均较低，且各亚种中出现的单元型散布在不同的进化枝中。结果表明SANDX-2位点的单元型在中国野猪中未形成明显的地理分布格局。若不考虑碱基的插入／缺失，用TCS构建19种单元型网络表明，单元型HAP2可能为一古老单元型，其他为衍生单元型。 2、研究优化了PCR-SSCP分型条件后，采用PCR-SSCP方法对中国野猪5个地方种群的41个个体的SLA-DQB外显子2进行分型。PCR-SSCP分型后共获得8个等位基因，发现15种标记带型，分别为AA、AB、BB、AC、AE、AH、BD、BF、BG、BH、DD、DG、DH、GG、EE。在鄂伦春地方种群中有6种带型，分别为AB，AE，AA，AC，EE和BB；红星地方种群中有4种带型，分别为AB，DH，BH和BB；古县地方种群中有3种带型，分别为AB，BB和AA；宝鸡地方种群有7种带型，分别为BB，BD，BF，BG，DG，GG和DD；金寨地方种群有3种带型，分别为AB，BH和AH。在8个等位基因中，等位基因B广泛分布在各个地理种群中。在中国野猪中，SLA-DQB外显子2等位基因的平均期望杂合度为0.6127，在各地方种群的平均期望杂合度在0.4444～0.7031之间。Hardy-Weinberg平衡分析表明SLA-DQB外显子2等位基因在野猪的5个地方种群中均未达平衡，这可能同该位点受平衡选择有关。地方种群间系统发生分析表明，鄂伦春种群和金寨种群彼此间的关系较近，红星种群、古县种群和宝鸡种群间的关系较近。更多还原

【Abstract】 The wild boar (Sus scrofa), which inhabited in wide areas of Asia, Europe, and North Africa, is an ancestral Species of domestic pigs. In this paper, a review of single copy nuclear DNA (scnDNA) analyses in population genetic studies was given. Based on the analysis of SANDX-2 sequence and SLA-SQB PCR-SSCP pattern, the genetic variations of the Chinese wild boars were discussed.1. The anonymous nuclear DNA SANDX-2 locus of 89 individuals of Chinese wild boars and a domestic pig were amplified and sequenced for studying the genetic variability of Chinese wild boars. Total of 510 bp were obtained after aligned, including 4 indels and 30 substitutions. Eighteen haplotypes were defined in Chinese wild boars, among which 7 haplotypes were observed only in one local wild boar population, and 11 haplotypes were shared by two or more populations. The phylogenetic tree constructed by the neighbor-joining method based on Kimura 2-parameters model divided the haplotypes into two distinct clades, and it was not supported by high bootstrap value. The results indicated there was no significant geographical distribution pattern in the Chinese wild boars. Ignoring the gaps, the nesting cladogram of 19 haplotypes suggested that HAP2 may be ancestral haplotype.2. With the optimized PCR-SSCP protocol, exon 2 of SLA-DQB gene of 5 local wild boar populations was genotyped by PCR-SSCP methods. Total of 15 genotypes (AA, AB, BB, AC, AE, AH, BD, BF, BG, BH, DD, DG, DH, GG, EE) were observed in 5 local populations. There are 6 types (AB, AE, AA, AC, EE and BB) in Elunchun population, 4 types (AB, DH, BH and BB) in Hongxing population, 3 types (AB, BB and AA) in Guxian population, 7 types (BB, BD, BF, BG, DG, GG and DD) in Baoji population and 3 types (AB, BH and AH) in Jinzhai population. Gene B was shared by all the populations. Expected heterozygosity of the DQB alleles in each population ranged from 0.4444 to 0.7031, and the average was 0.6127. Our results also show that the exon 2 of SLA-DQB departure from Hardy-Weinberg Equilibrium in all wild boar populations, this may correlate with balancing selection in the MHC DQB alleles. Phylogenetic analysis indicated that there were closer relationship between Elunchun population and Jinzhai population, and among Hongxing population, Guxian population and Baoji population.更多还原