基于微卫星DNA和线粒体COI基因对辽宁地区东北林蛙遗传多样性的研究

发布时间：2018-03-04 18:16

本文选题：微卫星　切入点：线粒体COI基因　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：东北林蛙是主要分布于我国东北地区长白山脉的两栖动物。近年来由于对东北林蛙的需求逐年上涨,野生东北林蛙资源就显得弥足珍贵。辽宁地区对东北林蛙的人工养殖也已广泛开展,为了不使人工养殖的过程中的引种及养殖方式对野生群体产生影响,研究东北林蛙遗传的多样性和遗传结构,以及人工养殖林蛙与野生林蛙的遗传多样性的差异就变得非常重要,而分子标记正是开展这项工作的理想手段。本研究分别利用核基因组微卫星标记(SSR)和线粒体DNA(mtDNA)的COI基因,研究了辽宁抚顺、本溪、铁岭、丹东地区东北林蛙群体,共171个个体的遗传多样性和遗传结构。(1)本研究选择了能够稳定扩增的多态微卫星位点8个。在这8个多态位点中,位点Rpi100在抚顺群体、本溪群体、铁岭群体中表现为单态,在丹东群体中表现为多态。8个多态性位点的多态信息含量平均值为0.5188,在4个东北林蛙群体171个样本中总共检测到87个等位基因。各位点的等位基因数为3～29,平均等位基因数为10.88,平均有效等位基因数(A)为4.5989,平均观测杂合度(Ho)为0.2842,平均期望杂合度(HE)为0.6475。丹东群体的HE最高,为0.5718;抚顺群体的HE最低,为0.4821。同时观测到大量稀有等位基因,并且大部分稀有等位基因的频率都较低。4个群体中,只有位点Rpi104符合哈代-温伯格平衡(P0.05),其余位点均显著偏离哈代-温伯格平衡(P0.01)。(2)本研究获得了东北林蛙的线粒体COI基因部分序列,序列长度为630bp,所测得的所有序列T、C、A和G的含量分别为26.9%、30.4%、24.9%和17.8%,转换/颠换值为12.8,其中变异位点数为95个,多态信息位点数为13个。各群体的单倍型数(nh)共24个,单倍型多样性(π)为0.414±0.124～0.846士0.041。除丹东群体外,其他群体的Tajima'sD中性检验值变化范围为-1.4676～-1.0521,均未显示出显著性(P0.10),与中性突变相符。(3)基于微卫星DNA的分析结果显示,4个东北林蛙群体之间的分化极显著(FST0.25)。利用邻接法(Neighbour-Joining),分别基于微卫星DNA和线粒体COI基因重建了系统发育树,结果均显示本溪群体与其它群体遗传距离较远,但并未产生物种级别的分化。基于微卫星DNA和线粒体COI序列分析比较了群体间的遗传分化,AMOVA结果均表明遗传差异主要来自群体内,比例分别为71.85%和84.33%。(4)对铁岭人工养殖群体与其他三个野生群体进行了遗传多样性比较,结果显示人工养殖群体与野生群体的遗传多样性水平相近,并且有部分遗传多样性指标为4个群体中最高,说明通过封山封沟进行的人工养殖方式对于东北林蛙的遗传多样性未见负面影响;但铁岭群体与其它群体间的基因流较低,可能需要视情况补充野生个体,以维持群体的遗传多样性水平。本研究结果分别研究了野生状态下和人工养殖状态下的东北林蛙遗传多样性以及种群分化水平,不仅为保护野生群体的遗传多样性提供理论依据,也为东北林蛙的人工养殖方式提供理论支持。
[Abstract]:Northeast forest frog is mainly distributed in the Changbai Mountains in the northeast of China. In recent years, due to the amphibious animal r.dybowskii demand rose year by year, the wild Rana dybowskii resources is precious. Liaoning area of artificial breeding of Rana dybowskii has been widely carried out, in order not to make the introduction and breeding process. The impact on the breeding of wild populations, diversity and genetic structure of r.dybowskii genetic differences, genetic diversity and artificial breeding of Rana chensinensis and wild sex becomes very important, and the molecular marker is the ideal means to carry out the work. In this study, using nuclear microsatellites mark (SSR) and mitochondrial DNA (mtDNA) COI gene of Liaoning, Fushun, Benxi, Tieling, the northeast forest frog population in Dandong area, a total of 171 diverse individual genetic and genetic structure. (1) this study chose to Polymorphic microsatellite loci were 8. In the 8 polymorphic loci, Rpi100 loci in Fushun population, Benxi population, Tieling population showed a single state, in the Dandong group for the polymorphism information content of polymorphic.8 polymorphism loci with an average of 0.5188, 4 in the northeast forest frog population 171 a sample of a total of 87 alleles were detected. The number of alleles at all loci was 3~29, the average number of alleles was 10.88, the average effective number of alleles (A) was 4.5989, the average heterozygosity (Ho) was 0.2842, the average expected heterozygosity (HE) of 0.6475. group in Dandong the highest HE, 0.5718; Fushun group has the lowest HE, 0.4821. also observed a large number of rare alleles, and most of the frequency of rare alleles were lower than.4 group, only the site of Rpi104 with Hardy - Weinberg equilibrium (P0.05), the remaining loci were significantly deviated from the Hardy temperature Berg equilibrium (P0.01). (2) this study was part of the mitochondrial COI gene sequence r.dybowskii, sequence length of 630bp, measured by all the sequence T, the C, the content of A and G were 26.9%, 30.4%, 24.9% and 17.8%, the transition / transversion value is 12.8, the variant the number is 95, the polymorphic information loci was 13. Each group of haplotypes (NH) a total of 24, haplotype diversity (PI) was 0.414 + 0.124 to 0.846 + 0.041. group in addition to Dandong, other groups Tajima'sD neutral test value range is -1.4676 ~ -1.0521, showed no significant (of P0.10), consistent with the neutral mutation. (3) microsatellite analysis results revealed that the DNA based on the differentiation between the 4 groups of r.dybowskii significantly (FST0.25). By using the neighbor joining method (Neighbour-Joining), respectively DNA microsatellite and mitochondrial COI gene based on the reconstruction of the phylogeny tree, results showed that Benxi group and other groups Genetic distance, but did not differentiate species level. DNA microsatellite and mitochondrial COI sequence analysis and comparison of genetic differentiation among populations based on AMOVA. The results show that the genetic variation mainly within populations, the ratio was 71.85% and 84.33%. (4) of the Tieling artificial breeding group and other three wild populations comparison of genetic diversity, the results showed similar levels of genetic diversity of cultured and wild populations, and some genetic diversity index was the highest in 4 groups, that by blocking the channel of the artificial breeding method for genetic diversity of r.dybowskii no negative effect; but the population of Tieling and other the gene flow is relatively low, as the case may need to supplement wild individuals, in order to maintain the diversity of population genetics. The results of this study were studied under the condition of wild and artificial breeding. The genetic diversity and population differentiation level of Rana chensinensis under the condition not only provide theoretical basis for protecting the genetic diversity of wild populations, but also provide theoretical support for artificial breeding of Rana chensinensis.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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