【作者】 闫小玲；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2010， 博士

【摘要】 银杏(Ginkgo biloba L.)是第三纪孑遗植物,世界著名的“活化石”。关于银杏的冰期避难所、群体遗传多样性和遗传结构一直以来都是植物学家们感兴趣的话题。在前期研究的基础上,本研究通过单亲遗传的cpDNA基因间隔区序列变异以及双亲遗传的核基因微卫星位点(SSR)分析,对国内外银杏群体进行了深入的谱系地理学和群体遗传学研究,探讨了中国银杏的群体遗传结构和遗传多样性,在银杏避难所群体生存现状的基础上,提出了相应的保护策略。主要研究结果如下：1.cpDNA trnK基因,trnS-trnG基因间隔区和atpH-atpI基因间隔区序列分析在对33个群体430个个体的分析中,cpDNA trnK基因,trnS-trnG和atpH-atpl基因间隔区序列共检测到15种单倍型。银杏物种水平的单倍型多态性(h)为0.352,核苷酸多态性(π)为0.580×10-3,遗传多样性水平总体较低。西南地区和东部TM群体的遗传多样性较高。分子差异分析显示遗传变异主要存在群体内(55.50%),但群体间已经出现相当程度的遗传分化(44.50%)。贝叶斯分析和单倍型分布揭示了中国的西南地区和东部地区形成两个不同的谱系,表明中国西南地区和中东部地区的银杏形成了2个显著进化单元,是银杏的冰期避难所。2.SSR标记的筛选采用磁珠富集法开发了11对银杏微卫星引物。11个位点在40个个体中共得到100个等位基因,杂合度的期望值(HE)和观察值(Ho)的平均值分别为0.795和0.484,多态信息位点(PIC)平均值为0.752,表明这些位点具有较高的多态性。上述结果为全面深入研究银杏群体遗传和谱系地理打下了坚实的基础。3.SSR分析首次运用14对SSR标记分析了银杏的群体遗传结构和遗传多样性(33个银杏群体共980个个体),结果揭示出中国中部群体的遗传多样性较高,可能是种质多样和群体的混杂造成的。银杏群体的遗传变异主要存在于群体内(88.10%),群体间的遗传分化Fst= 0.119。Structure分析揭示的结果与cpDNA相同,即西南地区群体和东部地区群体呈现出两个独立进化的路线,东部的天目山、西南的WC群体和JF群体具有较高的遗传多样性和稀有等位基因丰富度,而中部群体则表现出东、西群体基因库混合的状态。结合cpDNA数据,我们进一步确定了中国西南地区的贵州务川和重庆金佛山以及东部浙江西天目山地区为银杏的冰期避难所。韩国栽培群体表现出与中国西南群体和中部群体较高的相似性,日本栽培群体则与中国中东部地区相似。欧美古老的银杏大树来自于日本东京群体,欧美其余个体与中国、韩国和日本群体之间均出现了显著的遗传分化。4.银杏的进化历史和传播历史基于BEAST运算得到这两个谱系最近的共同祖先距今约1.9(1.2-2.8)百万年,当时正处于第三纪末第四纪初,因此为古老的避难所。在距今约70万年前的间冰期,中国中东部银杏群体经历了快速的扩张。基于SSR分析的稀有等位基因丰富度表明,离避难所越远,群体的稀有等位基因丰富度逐渐下降,说明冰期后银杏从就近的避难所开始向外扩张。中国中部地区则成了两个避难所群体基因库的混合区域。银杏有着非常悠久的栽培历史,SSR分析揭示出银杏各栽培群体的来源不同,遗传多样性低的群体其来源比较单一。韩国与日本群体的遗传多样性均较高,说明其是从中国的多个地方多次引入的。欧美的银杏遗传多样性程度都很低,并且与其他群体出现了明显的遗传分化。银杏花粉流与种子流的比值为4,显著低于其它风媒传粉且繁育系统为异交的植物,表明人类活动对银杏基因流尤其是种子流的影响非常大,也可能是人类活动促进了银杏群体间的基因交流,使其度过灾难存活至今。5保护策略通过对避难所银杏群体和栽培群体的遗传结构和遗传多样性比较及两个避难所群体间遗传多样性的分析,提出以下保护策略：(1)应在中国西南地区,尤其是在贵州务川(WC)建立银杏保护区,对务川县山江村的银杏古树应该重点保护；(2)浙江西天目山的银杏群体具有较高的遗传多样性,在迁地保护上具有优先权。更多还原

【Abstract】 Ginkgo biloba L., one of the Tertiary relict plants, was referred to as the living fossil and a widely cultivated plant. The questions about the possible wild populations and evolutionary history of Ginkgo are crucial to understand the historical processes which Ginkgo had undergone. Now we studied on the population genetic structure and phylogeography of G. biloba including 33 populations from China and samples of cultivated populations from Korea, Japan, Europe and North America. This work was carried out based on cpDNA trnK gene and trnS-trnG and atpH-atpl intergenic spacer,14 pairs of SSR loci to investigate the genetic diversity and the glacial refugia and patterns of population genetic structure at the scale of China. There are some main conclusions of our research:1. cpDNA trnK gene, trnS-trnG and atpH-atpl intergenic spacer sequencesThirty-three populations (430 individuals) were analyzed using cpDNA trnK, trnS-trnG and atpH-atpl intergenic spacer. A total of 15 cpDNA haplotypes were detected. Nucleotide diversity and haplotype diversity areπ=0.580×10-3 and h= 0.352 respectively. SW China and Eastern China had higher genetic diversity. AMOVA revealed 55.50% variation exist within populations. High genetic differentiation was detected (Fst=0.44506) among populations.Phylogenetic analysis and nested clade analysis of cpDNA haplotypes revealed that two refugia existed during the glaciation:the first was located in SW China and the second in Mt. TM in Eastern China. The present populations derive their genetic make-up from two inferred clusters.2. Development of SSR markersWe isolated and characterized 11 polymorphic microsatellite loci from G. biloba using a modified biotin-capture method. These loci showed high allelic diversity with PIC ranging from 0.432 to 0.909 (average= 0.752). The observed and expected heterozygosities ranged from 0.208 to 0.708 (average= 0.484) and from 0.501 to 0.934 (average= 0.795), respectively. These microsatellite markers can be used as genetic markers for further studies on genetic diversity and the gene flow among populations of this species.3. SSR analysisA total of 980 individuals from 33 populations in China, Korea, Japan, Europe and North America were investigated using 14 pairs of SSR loci. High genetic diversity was detected in WC (0.741), XA (0.810) and TM (0.788). Low genetic differentiation among populations was detected with Fst= 0.119. Structure analysis revealed two major phylogeographical groups in China:SW China, Eastern China and the mixture of other populations. This pattern probably reflected the isolation of the SW populations and Eastern populations have independent evolution history.Thus, SSR analysis combined cpDNA data revealed two refugia areas. European samples are genetically divergent from Chinese populations, indicating their indirect origin from China, but it is more closely related to the Eastern populations in China.4. Evolutionary and cultivation historycpDN A haplotypes and SSR analysis had certained two glacial refugia of G. biloba during the interglacial period. The phylogeny tree based on Bayesian method showed there were two lineages in G. biloba. The posterior probability of these two lineages was 1 and indicated that two significant evolutionary unit had been formed. BEAST showed the most recent common ancestor of the two lineages was 1.9 million years ago. Mismatch distribution supported significant postglacial population expansion in Eastern China in 701 kya BP, the southwest region did not experience significant expansion, but there are some outside groups in the refugia undergone a short-range diffusion. The rare allelic richness based on SSR analysis showed that the farther away from the glacial refugia, the rare allelic richness decreased, indicating the postglacial recolonization from the nearest refugia. Central China was the secondary contact zone of both glacial refugia.Ginkgo has a very long cultivation history in Eastern Asian. SSR analysis revealed that some cultivation population had different sources. Korean population showed the similarity with central and southwest China, while the Japanese populations were similar to Central and Eastern China, the higher genetic diversity indicated that they might be introduced from China for many times and from various locations. Genetic diversity in Europe and North America are very low. As the wind pollination of woody plants, the seeds of G. biloba hadn’t the structure of long-distance dispersal, but the ratio of pollen flow/seed flow was 4 and lower than other wind pollination plants. This may indicate human activities promoted gene flow among Ginkgo populations.5. Conservation strategiesThe genetic structure detected in this study suggested some ideas for the management of G. biloba populations, especially for Southwest populations, a natural reserve must be established to protect the ancestral refugia in this area. In contrast, the population in Mt. Tianmu should have the highest conservation priority in ex situ conservation. In some cultivated populations and marginal populations, the low genetic diversity requires careful consideration.更多还原