【作者】 高嵩；

【导师】 张恒庆；

【作者基本信息】 辽宁师范大学 ， 植物学， 2009， 硕士

【摘要】 本研究选取了我国东北部吉林、辽宁、黑龙江三省25个红松种群共125个个体为研究对象,研究红松线粒体DNA coxIII片段的遗传多样性及红松种群的亲缘关系。研究选用coxIII引物片段为5’CAGTACCATGCAGCTGCTTC3’, 5’GGTAGATCCAAGTCCATGGC3’。经过模板DNA提取、模板DNA纯化、PCR扩增、PCR产物鉴定、PCR产物测序、测序结果分析等实验过程,得到结论如下:1.红松线粒体DNA变异。本研究主要从核苷酸组成、coxIII片段长度、核苷酸变异三个方面研究红松线粒体DNA遗传多样性。核苷酸组成方面,经过分析四种核苷酸平均值及变异范围为:A 21.7%(21.4%-22.2%);T 30.9%(30.5%-31.7%);G 22.4%(22.2%-22.6%);C 25.1%(24.7%-25.2%),coxIII片段长度范围在628bp-631bp之间,共4种长度序列,片段长度变异程度很小。经过Clustal软件分析发现30个变异位点,共变异68次,变异位点比率为4.7%,其中单一多态位点23个,简约信息位点7个。通过以上数据可以看出红松线粒体DNA变异在线粒体DNA变异中水平较高。2.红松种群的单倍型。本研究对所选样品种群的单倍型进行了检测,共发现36种单倍型,命名为H1-H36,其中有一种(H1)为主流单倍型,推测出现主流单倍型的原因为线粒体DNA变异速率很小导致的。在所有种群中都存在。另外35种单倍型分布在19个种群中,文中介绍了这35个单倍型在这19个种群中存在情况。3.系统进化树的构建。本研究应用MEGA 4.0软件中的邻位相连法(Neighbor-Joining)和最小进化法(minimum evolution),以红松种群中各单倍型为对象分别构建NJ进化树和ME进化树。通过构建系统进化树,找到了红松种群间的进化亲缘关系,H13、H14、H15、H21、H22、H24、H26、H27、H29、H31、H32、H33相聚,说明亲缘关系较近,而H23和H10又和他们关系较近;而H16、H11、H3之间聚类到一起,证明亲缘关系较近;H4、H5、H6、H7、H8、H17、H30等聚类的一起,证明他们亲缘关系较近。因红松线粒体为父系遗传,所以亲缘关系相近种群间存在花粉流动。分析花粉流动可能原因为山脉阻隔和气候影响。在国内外研究中,对红松种群亲缘关系的研究很少,而且普遍的观点认为线粒体DNA序列较保守,不适合用于植物亲缘关系研究。而红松线粒体为父系遗传,对其基因组序列的研究有着特殊意义。本研究不仅为今后研究红松进化提供了科学参考,而且为红松资源保护提供理论依据。更多还原

【Abstract】 This study select koraiensis 25 populations for a total of 125 research subjects from the north-east of China-Jilin, Liaoning, Heilongjiang, research fragment of mtDNA pinus koraiensis coxIII genetic diversity and phylogent evolution.Study select coxIII’s primer fragments are 5’CAGTACCATGCAGCTGCTTC3’, 5’GGTAGATCCAAGTCCATGGC3’. Through extraction of template DNA,template DNA purification, PCR amplification, PCR product identification, PCR product sequencing, sequence analysis, the results of the experiment have been concluded as follows:1. Analysis of pinus koraiensis mtDNA variation. We study the nucleotide variation of mtDNA genetic diversity of pinus koraiensis from the nucleotide composition, coxIII fragment length, mtDNA variation. Through analysis of nucleotide composition ,the ranges of the four nucleotides and the average are that A 21.7% (21.4% -22.2%); T 30.9% (30.5% -31.7%); G 22.4% (22.2% -22.6%); C 25.1% (24.7% -25.2%).there are some variations, but the degree of variation was not obvious, and less individual Mutated. Range of coxIII Fragment`s length is 628bp-631bp, four kinds of length sequences, the degree of fragment length variation is very little. After the Clustal analysis we get 30 Variable sites, the times of the total variation are 68 ,the rate of Variable sites is 4.7%, the singleton variable sites are 23, parsimony informative sites 7, the degree of pinus koraiensis mtDNA mutate is higher.2. determine the haplotypes of Koraiensis populations. In this study, samples of the populations selected are found 36 kinds of haplotypes named H1-H36, in which there is one kind haplotype (H1) for the mainstream haplotype in all populations exist. 35 haplotypes exist in 19 populations, this article detailed describe the Relations 19 populations and 35 haplotypes.3. Construction of phylogenetic trees. In this study, using the MEGA 4.0 software Neighbor-Joining and minimum evolution method, in the target of pinus koraiensis populations haplotypes, phylogenetic trees were constructed. By constructing phylogenetic trees we found the evolution relationship of pinus koraiensis populations, H13, H14, H15, H21, H22, H24, H26, H27, H29, H31, H32, H33 have closer relationship, H23,H10 are closer with them; and H16, H11, H3 clustering together prove that they have closer genetic relationship; H4, H5, H6, H7, H8, H17, H30 such as clustering together to prove that they have closer genetic relationship. mtDNA have paternal genetic effect,there is Pollen flow between closer relationship populations.the possible reasons are the mountain barrier and climate.Among the Study home and abroad, there are few research about the pinus koraiensis populations Relationship and at the most time researcher think that mtDNA sequence is too Conservative to the study of plant relationships. For the male Genetic of the pinus koraiensis, mtDNA sequence has a special significance to the study. this study not only provide scientific future research not only provides scientific reference for future research, but also provide the theoretical basis for the pinus koraiensis hybrid and pinus koraiensis breeding.更多还原