【作者】 杨雪；

【导师】 赵桂仿； 刘占林；

【作者基本信息】 西北大学 ， 植物学， 2007， 硕士

【副题名】基于cpSSR和AFLP标记

【摘要】 巴山松(Pinus henryi Mast．)是我国秦巴山区特有种，主要分布于大巴山脉、巫山支脉以及云贵高原延伸的余脉上，但数量稀少，已被列为珍稀植物。分类上巴山松常作为油松或马尾松变种处理，个别专家将其认作独立种。在地理分布上，巴山松北与形态难以区分的油松相连，东与容易混淆的黄山松比邻，南与云南松交错，又与马尾松重叠分布(马尾松分布海拔更低)。形态特征的相似、分布的重叠以及杂交现象的存在都说明巴山松及其近缘种存在目前尚未清楚的复杂的种间关系。本研究从分子系统学角度出发，运用cpSSR(叶绿体微卫星)和AFLP两种分子标记技术对巴山松及其近缘物种黄山松、马尾松、油松和云南松等五种松树的遗传多样性、遗传分化和系统发育关系进行分析，探讨该五种松树复杂的种间关系，为科学地保护和利用我国的特有的松树资源提供理论参考。研究结果表明：(1)五种松树都表现出较高水平的遗传多样性与种间分化。但cpSSR比AFLP标记提供了更多的种内种间分化，而展示的遗传多样性相对要少。在对不同的方法指标对比研究后，我们认为将cpSSR的单倍型关系考虑在内，能够更有效地分析植物居群遗传分化造成的地理结构，但解释时必须谨慎。而对于显性标记AFLP，传统的平方根法会低估遗传参数，而Lynch和Milligen的方法因样本量的限制也可能高估遗传多样性，Bayesian法被认为是最精确的方法，提供了与分子方差分析(AMOVA)近似的遗传分化值。(2)巴山松作为一个独立的种是成立的，并且巴山松与黄山松亲缘关系最近。进一步分析表明，巴山松与黄山松、云南松与油松分别向着两个方向分化，马尾松拥有更多祖先基因组成分，占据着中间类型。而个体聚类图则显示马尾松、黄山松、云南松和油松这四种松树种间关系极为复杂，难以区分。松属植物这种种间关系不清的现象可能与分化时间较晚以及频繁的种间杂交有关。另外，本研究以巴山松及其近缘种为例，利用cpSSR和AFLP两种分子标记对其取样策略和统计方法进行分析，揭示它们对遗传多样性和亲缘关系确定的影响。结果表明，居群取样个体数和基因位点数差异对遗传多样性影响并不显著，但它们和系统树构建方法对近缘种亲缘关系的确定有一定的影响。更多还原

【Abstract】 Pinus henryi Mast., an endemic species in QinBa mountains of China, mainly distributes over Daba mountains, Wushan mountains and offshoots of YunGui plateau. Most forest of Pinus henryi have been destroyed by man and this species is listed as rare plant.Pinus henryi is often regarded as taxonomic variant species of P. tabuliformis or P. massoniana. Several experts argued that it should be independent species based on the differences of timber anatomy and chemical components. According to geographic distribution, P. henryi is linked together with P. tabuliformis in north, neighbor with P. taiwanensis in east, crisscross with P. yunnanensis in south, and overlap with P. massoniana (lower at altitude). Complicated relationships exist in Pinus henryi and its relative species due to similar morphic characteristics, overlapped distribution and extended hybridization in pines. Making use of amplified fragment length polymorphism (AFLP) and chloroplast simple-sequence repeat (cpSSR) markers, we analyzed molecular Phylogenetics, gene diversity and genetic differentiation in these five Eurasian pines. The study would identify phylogenetic relationship of P. henryi and its relative species, and imply for protecting and utilizing the special pine resources in China.The results were as follows: Firstly, high genetic differentiation and median gene diversity with cpSSRs markers were found both at population and species level while median differentiation and higher gene diversity with AFLPs data. Although the Infinite Allele Model were preferred at most chloroplast gene loci, other models should not be rejected according to the similar support rates. Measures of subdivision that consider similarity between haplotypes offered better information of plant geographic structure than the standard ones. Among several methods analyzed in AFLPs, Sequare root method provided downwardly biased estimates of the genetic parameters, while Lynch and Milligen method over-estimated genetic diversity due to limited samples. Thus Bayesian statistics was the most accurate and popular method in those data with the dominant markers and its value of species differentiation (θ~B = 0.1035 ) was close to the parameter given by Analysis of Molecular Variance (AMOVA).Secondly, it was evident that P. henryi was an independent species and close to P. taiwanensis using both cpSSRs and AFLPs data with high bootstrapping supports. P. henryi and its relatives shared more common genetic components given by AFLPs than those offered by cpSSRs results, which suggested that late divergent time or frequent gene flow might contribute to phenomena of the shared genomes.In addition, taking P. henryi and its relative species for example, sampling strategy and statistical methods were analyzed by chloroplast microsatellites (cpSSR) and AFLP markers. The results showed some differences with present views. It was phylogenetic relationships rather than genetic diversity that was notablely affected by sample sizes/loci and methods of phylogenetic trees construction.更多还原