石河子大学 硕士学位论文 准噶尔雅罗鱼遗传多样性及其同域同属种间基因渐渗研究 姓名：涂著池 申请学位级别：硕士 专业：遗传学 指导教师：胡文革 2011-06 I 摘 要 准噶尔雅罗鱼(Leuciscus merzbacheri)属鲤形目(Cypriniformes) 鲤科(Cyprinidae)雅罗鱼亚科 (Leuciscinae) 雅罗鱼属（Leuciscus cuvier ） 又名新疆雅罗鱼，仅分布于新疆准噶尔盆地，准噶尔雅 罗鱼是我国新疆特有的土著鱼类物种，也是该地区的鱼类区系中最典型的代表种由于近年来的不 合理开发，造成这一珍贵鱼种种质资源急剧下降，面临灭绝的危险，已被列为新疆濒危野生鱼类物 种然而，有关准噶尔雅罗鱼 D N A 分子水平遗传多样性方面的研究还属空白，尚未见有人报道，因 此，对准噶尔雅罗鱼分子遗传多样性的研究显得刻不容缓针对这一情况，本试验以分布于新疆准 噶尔盆地的准噶尔雅罗鱼以及分布在同一水域- - 赛里木湖中的贝加尔雅罗鱼和高体雅罗鱼为试验材 料，使用 I S S R 分子标记技术，研究准噶尔雅罗鱼的遗传多样性，并探讨共同栖息于赛里木湖的三种 雅罗鱼亚种间是否发生基因渐渗。
本试验以采集到的准噶尔雅罗鱼 D N A 样本为模板，利用优化的 PCR 体系从 32 条引物中筛选得 到的 9 条可用于本研究的 ISSR 引物，并确定其在这一体系下各引物的退火温度 将采集到的准噶尔 雅罗鱼（3 个群体分别来自赛里木湖 sz、精河 jz、奎屯河 kz） 、贝加尔雅罗鱼（1 个群体来自赛里木 湖 sb） 、高体雅罗鱼（2 个群体分别来自赛里木湖 sg 和额尔齐斯河北屯河段 bg）6 个群体共 91 个样 本， 分别提取 DNA 样本， 并以其 DNA 样本为模板进行 PCR 扩增， 6 个群体 91 个样本通过 9 条 ISSR 引物共扩增出 193 条带，片段长度在 100~2000 之间，其中多态性条带为 178 条，6 个雅罗鱼群体的 多态性位点比率（PPB）为 91.75%，表明整个雅罗鱼群体的遗传多样性水平很高分析每个群体的 多态位点水平，6 个群体 PPB范围：9.28%~47.94%，水平较低，群体间的遗传多样性水平高低依次 是 bgszjzsbkzsg 将电泳图谱中清晰的条带 （包括弱带） 记为 “1” ， 否则记为“0” ， 建立 0/1 数据矩阵。
POPGENE3.2 软件分析群体内遗传距离（Dc） 、 基因多样性指数（Nei） 、 Shannon 信息指数（I） 、 遗传分化系数 Gst、 基因流（Nm）等遗传多样性数据结果表明：在物种水平上，遗传多样性高（PPB 91.75%, H 0.3352, I 0.4985） ，而群体内的遗传多样性水平较低；遗传分化水平较高（Gst=0.7039） ，基因流水平较低 （Nm=0.2103） ，平均遗传距离为 Dc=0.4594 利用 Arlequin3.5 软件作分子差异（ AMOVA）分析，结果显示 45.36%的分子遗传变异来自种间， 30%的遗传变异来自群体间，而群体内部的遗传变异仅占 24.64%物种间的遗传变异大于群体及群 体内部的遗传变异用聚类分析软件 NTSYS- pc 构建聚类图三种雅罗鱼 6 个群体大体聚为两个大 类群，准噶尔雅罗鱼和贝加尔雅罗鱼聚为一个类群，高体雅罗鱼聚为一个类群，而在准噶尔雅罗鱼 一簇中又聚为赛里木湖的 sz、sb和 jz、kz 两个亚类群结果表明，赛里木湖准噶尔雅罗鱼与贝加尔 雅罗鱼之间存在一定程度的基因交流，有发生基因渐渗的可能；而准噶尔雅罗鱼与高体雅罗鱼及贝 加尔雅罗鱼与高体雅罗鱼之间缺乏基因交流。
说明准噶尔雅罗鱼比其他两种雅罗鱼表现有较强的环 境适应能力和进化潜力 本文采用 ISSR 分子标记技术为手段，分析了准噶尔雅罗鱼的遗传多样性水平，对遗传多态性研 究可以反映物种或群体的进化历史，分析其进化潜力，还有助于对物种稀有或濒危原因及过程进行 研究；而开展准噶尔雅罗鱼与其同属鱼种之间基因渐渗方面的研究工作，将为准噶尔雅罗鱼细胞遗 传学和比较基因组学提供珍贵的背景资料，对于确定鱼类的分类地位及演化关系打下基础也为该种 资源保护提供科学依据 关键词：准噶尔雅罗鱼，I S S R ，遗传多样性，基因渐渗 II Abstract Leuciscus merzbacheri (Zugmayer) is a genus of fish belonging to Cypriniformes，Cyprinidae， Leuciscinae，Leuciscus cuvier. Zugmayer is an indigenous medium- sized fresh or brackish fish and other two Leuciscus species (L. baicalensis and L.idus), distributed over Junggar Basin of north- region of Xingjiang, Northwest China. Resently, most populations all over drainages suffered severe demographic decline and habitat fragmentation due to unreasonable development and utilization. Thus, early in 1998, the native fish species have been listed as vulnerable species in the China Red Date Book of E ndangered Animals and it was listed as Xinjiang Aquatic wildlife protection list in 2004. However, limited genetic information is available for this rare dace, especially at molecule level. So it is imminent to launch this work. Here we describe the genetic diversity and introgression among Leuciscus Subspecies in Sailimu Lake by inter- simple sequence repeat markers (ISSR). In order to investigate the optimal ISSR- PCR system for Leuciscus merzabcheri for the ISSR- PCR approach defected by its unstable working condition. So, at first phage, genome DNA of L.merzbacheri were used as template DNA and L9（34）orthogonal design was adopted to optimize ISSR amplification system, which involved four principal factors, Taq polymerase, Mg2+, dNTPs and Primers at three levels, respectively. Then, we gained the optimized ISSR- PCR system with 1×PCR buffer, 0.5U Taq DNA polymerase, 3.0 mM MgCl2, 250µM dNTPs, 0.75µM primer and 30 ng template DNA in total 25ul reaction volume. Furthermore， we screening amount of 9 primers from 32 primers and obtained their suitable anneal temperature observed this system. The results set a basic step to analysis the genetic diversity of the rarely fish species. Furthmore, the genetic diversity of 91 individuals from six wild populations (including three populations of L.merzbacheri, two populations of L.idus and one population of L.merzbacheri) in Junngr Basin was investigated. Using the nine primers, 193 unambiguous DNA fragments were generated with 178 (91.75%) being polymorphic, indicating a notable genetic variation at the species level. However, at the population level, a low level polymorphism with the percentage of polymorphic bands ranging from 9.28 to 47.94%. Under the assumption of Hardy- Weinberg equilibrium, the Nei’ s gene diversity (Hpop) ranged from 0.0384(SG) to 0.1615(BG) with an average of 0.0994 at the population level, whereas Nei’ s diversity index ( Ht) value is 0.3352 at species level. Shannon index ( Ipop) ranged from 0.0561(BG) to 0.2419 (SG), with an average of 0.1496 at the population level and at species level the Shannon index (It) value is 0.4985. Thirdly, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between species. As the ISSR markers revealed, 45.36% of the total genetic diversity resided at the intraspecies level among populations, whereas 30% and 24.64% were present at interspecies and within a population respectively. The Nei’ s Gst (0.7039) and gene flow among populations (Nm=0.2103) revealed seldom g。