1、摘 要多倍化是植物进化的重要机制，因而成为系统与进化植物学研究的核心内容。多倍体可以分为同源多倍体和异源多倍体，其形成的重要原因是不减数配子融合。现在知道三倍体可能在多倍体形成过程中起了重要的作用，而且很多多倍体是多元起源的，并且对多倍体形成后其基因组和基因表达的变化也有了初步的认识。多倍体复合体是多倍体产生的原初阶段，许多关于多倍体的认识是建立在对多倍体复合体不同细胞型的比较研究基础上的。绵枣儿（Scilla sinensis Merrill）多倍体复合体是风信子科（Hyacinthaceae）在东亚的唯一类群。该复合体含有两个分化明显的基因组，到目前已经发现 15 种整倍体细胞型以及大量非
2、整倍体细胞型。其地理分布已经清楚而且形态变异复杂，是研究植物多倍体进化问题的理想材料。分子系统学将分子生物学的方法应用到系统学的研究中，极大的促进了系统进化研究。叶绿体 DNA 非编码区序列变异较快，是研究低等级类群系统关系的有利工具；而且，叶绿体的单系遗传特性对于研究多倍体可能的亲本是很有用的。许多数学方法被用于分析 DNA 数据，而计算机技术的飞速发展也使得用这些数学方法处理大量的数据成为可能。本文分析了采自东亚的 39 个居群叶绿体 DNA 序列 trnL-trnF 基因间隔区，trnL 内含子区以及 rpL16 内含子区，用 UPGMA 法采用 p 距离模型构建了系统树，并根据单倍型之
3、间的碱基差异多少，结合单倍型的地理分布绘制了单倍型地理分布图。实验结果如下所述：1.三条序列组合后的总长为 1883bp，共有 43个碱基替代位点和 11 个位置的插入/缺失。AA 细胞型 16 个居群有 12 个单倍型，BB 细胞型 13 个居群有 5 个单倍型，AABB 细胞型 8 个居群中有 4 个单倍型。2.AA 和 BB 细胞型构成单系类群，其 bootstrap 支持率高达 99。3.AA 和 BB细胞型间存在明显分化，所有 AA 细胞型个体构成一个单系，而所有 BB 和AABB 细胞型构成另一个单系。 4.AA 细胞型个体之间分化很大，BB 细胞型和AABB 细胞型分化较小，在系
4、统树上形成多分支。根据以上的结果和前人的研究，我们探讨了绵枣儿多倍体复合体各细胞型间的系统发育关系和现代地理分布式样的形成，得出如下结论：1.AA 和 BB 具有最近的共同祖先，推测 BB 可能是更古老的细胞型。2.AA 细胞型各居群间遗传差异相对较大，而且这种差异与各单倍型地理分布距离存在正相关。该细胞型随第四纪以来气温下降而扩大其分布区。其中北川居群可能代表 AA 细胞型的原始类型，由此形成三条散布路线，一是向西沿川西进入滇西北；二是向东转而向北进入黑龙江；三是向东转而向南经华中进入华南。3. BB 细胞型各居群间的遗传差异较小，但大体上可以看出从北向南的散布方向。该细胞型随第四纪以来气温
5、下降分布区逐渐由北向南收缩。4.AABB 细胞型各居群间的遗传差异比 BB 稍大，其分布区间断分布，没有明显的规律。推测所有 AABB 居群的母系都是 BB。灵岩山 2 较早分化出来，可能是较古老的类型。AABB 由多个单倍型构成，这有可能是 AABB 多元起源的结果。关键词 绵枣儿；多倍体复合体；trnL-F；rpL16 内含子区；分子系统学。AbstractPolyploidy, a crucial mechanism of plant evolution is identified as the core of the research in the systematic and evo
6、lutionary botany. Polyploid is divided into autopolyploid and allopolyploid. According to the recent research results, it is shown that unreduced gametes are discovered to be a major mechanism of polyploid formation, in which triploids may play an important role. It is also found that many polyploid
7、s have multiple origins, and that numerous changes take place in genome and gene expression after polyploids arise. Polyploid complex is at the initial stage of polyploid evolution. The comparison of diploid and polyploid cytotypes of polyploid complex attributes to the knowledge of polyploid.Scilla
8、 sinensis polyploid complex the sole taxon of Hyacinthaceae in East Asia has two genomes, which indicate prominent differentiation. By far, 15 euploid and many aneuploid cytotypes are found in Scilla sinensis polyploid complex, and its cytogeographic distribution has been dipicted. Scilla sinensis s
9、erves as ideal material in the research of the evolution of plant polyploidization.Molecular methods are adopted, which attribute greatly to the phylogenetic and evolutionary research. Rapid variation of cpDNA sequences shows in noncoding regions, which may assist in studying the phylogenetic relati
10、on of low taxon. Besides, it exhibits a characteristic of maternal parent inheritance, which is considered as of great help in studying the potential parents of polyploid. Many mathematic methods with the vast advance of computer techniques make possible dealing large numbers of DNA data. In this di
11、ssertation cpDNA trnL-trnF spacer, trnL intron and rpL16 intron of Scilla sinensis from 39 populations in East Asia are sequenced. We construct phylogenetic trees by means of UPGMA and p-distance, and draw a map of haplotypes geographic distribution on the basis of the differences and distribution o
12、f haplotypes. The results of the experiment are shown as below: 1. Three sequences are combined into one with a total length of 1883bp after alignment, in which about 43 substitutions and 11 indel are found. There lie 12 haplotypes in 16 populations of AA cytotypes, 5 haplotypes in 13 populations of
13、 BB cytotypes, and 4 haplotypes in 8 populations of AABB cytotypes. 2. AA and BB cytotypes are monophyletic group with the high bootstrap support of 100%. 3. There are distinct differentiation between AA and BB cytotypes in that all of AA cytotypes belong to a monophyletic group, and all of BB and A
14、ABB cytotypes belong to another monophyletic group. 4. In contrast to AA cytotypes, which show pronounced differentiation among populations, BB and AABB cytotypes have little differentiation which form multiclades on the phylogenetic tree.Based on the results of early research and above experiment,
15、the phylogenetic relations of the major cytotypes and the formation of the pattern of modern geography distribution of Scilla sinensis polyploid complex are discussed in the dissertation. Conclusions are drawn as follow: 1. AA and BB have a closest ancestry, and BB is supported to be more ancient. 2
16、. The genetic differentiation among AA populations is greater than among BB populations, which positively relates to the geographic distribution of the haplotypes. With the decreasing temperature after Quaternary, AA cytotypes have extended their distribution. Beichuan population is likely to repres
17、ent the ancient type of AA cytotypes, from which three lines radiate: first goes west via west Sichuan into northwest Yunnan; second goes east and then north into Heilongjiang; third goes east and then south via the center of China into south China. 3. Despite that genetic differentiation among BB p
18、opulations is not clear, their distribution area can be perceived from north to south as a whole, and shrinks with the decreasing temperature after Quaternary. 4. A larger genetic differentiation is found among AABB populations than among BB. The distribution region of AABB cytotypes is discontinuou
19、s, which doesnt take regular pattern. The maternal of all AABB cytotypes are supposed to be BB. Lingyanshan-2 may be an ancient type of AABB. AABB cytotypes are made up of several haplotypes, which may results from their multiple origins.Key words Scilla sinensis; Polyploid complex; trnL-F; rpL16 in
20、tron; Molecular phylogenetics.目 录前言.1一.植物多倍体的研究进展.11.多倍体的类型.12.多倍体的形成途径.23.三倍体桥在多倍体产生过程中的作用.24.多倍体的多元起源.25.多倍体基因组的进化.36.多倍体新的基因表达机制.3二绵枣儿多倍体复合体的研究历史.41.绵枣儿的细胞型组成.42.A 和 B 基因组的关系.53.绵枣儿的现代细胞地理分布.6三分子系统学概述.71.rpL16，trnL 内含子区以及 trnLtrnF 基因间隔区概述82.系统发育分析的数学方法.9材料和方法10一.材料采集与总 DNA 提取纯化.101.材料采集和处理.102.总
21、DNA 的提取、纯化、鉴定.10二.目的片断 PCR 扩增111.PCR 反应所需引物的合成112.PCR 反应143.PCR 反应结果的检测14三序列测定.15四序列数据处理和系统树构建. 151.序列编辑、排序.152.序列数据分析.153.系统树构建.154.单倍型地理分布.16结果.16一序列变异的描述.16二系统树.161.绵枣儿及其近缘类群系统树. 162.绵枣儿各居群系统树17三.单倍型地理分布17讨论21一.AA 和 BB 细胞型具有最近的共同祖先22二.AA 和 BB 细胞型的分化22三.AA 细胞型居群之间的分化. 23四.BB 细胞型居群之间的分化. 24五.AABB 细胞型居群的起源和分化. 24六.绵枣儿多倍体复合体细胞型现代地理分布的形成25小结27参考文献27致谢33附表34
