摘要牛顿冷却定律
榕属(Ficus L.)植物数目众多，生活型多种多样，一些物种的形态特征不稳定，榕属植物与传粉小蜂之间存在的独特共生关系，都导致对榕属植物的分类鉴定存在很大困难，尤其是subsect. Frutescentiae中冠毛榕-异叶榕复合的分类问题。 本课题从60对SSR引物中筛选出了18对具有较高多态性的引物，通过荧光标记引物、定量扫描PCR产物片段长度，对冠毛榕-异叶榕复合内12个物种(共144份样本)进行了SSR分析，结果所有样本均成功扩增，每对引物扩增出6-22个等位基因，共获得268个等位基因，平均每对引物可扩增14.9个等位基因。SSR位点呈现较高的多态性(0.613<PIC<0.893)；聚类结果显示该复合内物种间的关系密切
(0.83<SM<0.97)。在此基础上获得以下结论和推论：
(1) 平塘榕(F. tuphapensis)与楔叶榕(F. trivia)聚在一起，说明二者关系密切，可以确定平塘榕应该是subsect. Frutescentiae (sect. Ficus)类的成员而不属于sect. Eriosycea。
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(2) 大明山榕(F. daimingshanensis)的所有样品嵌于异叶榕(F. heteromorpha)的一个分支中。推断大明山榕是异叶榕的一个变异类型，大明山榕的―种‖级地位不能成立。
(3) 冠毛榕(F. gasparriniana)的聚类情况非常复杂，共聚类为4个分支，其中一个分支与部分矮小天仙
果(F. erecta)聚在一起，二者再与台湾榕(F. formosana)构成妹；还有两个分支连同部分异叶榕聚在一起，最后一个分支与由沙坝榕(F. chapaensis)、异叶榕及大明山榕共同构成的分支构成妹关系。考虑到与矮小天仙果、台湾榕及沙坝榕相关联，冠毛榕与琴叶榕(F. pandurata)也相联系。可见这些物种之间也存在明显的基因渐渗或杂交现象。
(4) 石榕树(F. abelii)嵌于舶梨榕(F. pyriformis)所在分支中，形态解剖特征上的显著差异并未反映在SSR聚类树上，提示相关物种间可能存在近期发生的基因交流。
冠毛榕-异叶榕复合内这些物种间复杂的遗传关系，已经模糊了绝大部分物种间的界限，形态学上所划分的“种”，通常只是复杂网络关系中连续变异梯度
中的某一个点，并不能代表一个真实的生物学种。然而，要想明确复合内各生物学种之间的界限，仍需进一步的研究。 本研究为榕属植物复合研究提供了新思路，为更好的理解榕-榕小蜂之间互惠共生关系提供了分类学资料。亚里士多德柏拉图
关键词：SSR；复合；榕属；分类学；冠毛榕；异叶榕
Abstract
聚乙二醇醚
Species richness, life-form diversity, morphological character variablity, and unique symbiotic relationships between figs and their pollinating wasps, could account for the great difficulty in classifying and delimiting Ficus species (Moraceae), especially in F. gasparriniana - F. heteromorpha complex that belong to subsect. Frutescentiae (sect. Ficus)
Microsatellite analysis on 12 species, 144 samples belonging to F. gasparriniana - F. heteromorpha complex was carried out using 18 fluorescently labelled microsatellite primers (screened from 60 primers). The PCR products of each sample scanned quantitatively to gain fragment length. It shows that all samples were amplified successfully, a total of 268 allels were obtained and the number of alleles per locus varied from 6 to 22 with an average of 14.9. SSR loci reveal high polymorphism (0.613<PIC<0.893); cluster tree shows the intimate relationships among the complex species (0.83<SM<0.97). Conclusions and some deductions are listed as follows:
(1) F. tuphapensis clustered with F. trivia, indicates their intimate relationship, and F. tuphapensis belongs to subsect. Frutescentiae (sect. Ficus) rather than sect. Eriosycea.
(2) That all samples of F. daimingshanensis share one clade with some samples belonging to F. heteromorpha, infers F. daimingshanensis should not be treated as a species but as a variant type of F. heteromorpha.
(3) Clustering results of F. gasparriniana are complicatied, with four scattering clades. One clustered with F. erecta, then as a sister to F. formosana; two clades clustered with some samples of F. heteromorpha; and the forth clade clustered as a sister relationship with the branth, containing F. chapaensis, F. heteromorpha and F. daimingshanensis. Considered its relationship with F. formosana, F. chapaensis and F. erecta, F. gasparriniana also have relationship with F. pandurata. Introgression or hybridization may exsit among all these species.
(4) F. abeli embed in clade of F. pyriformis, not in accordance with their significant differences in morphological and anatomical characters, which reminders interspecific gene exchange may occur recently.
Complicated genetic relationships among F. gasparriniana- F. heteromorpha complex have already perplexed most species boundaries. ―Species‖determined by morphological characters usually just a ―dot‖belonging to continuous variation gradient of complicated relation network rather then a real biology species. However, how to make the boundaries among biology species in the complex clear need a further more research.
This study provides a new idea for complex studies of Ficus species, and also new taxonomic data on better understanding of the fig – fig wasps mutualistic system.
Keywords:SSR; complex; Ficus; Taxonomy;Ficus gasparriniana; Ficus heteromorpha
目录
1 榕属植物研究综述 (1)
1.1 榕属植物简介 (1)
1.2 榕属经典分类研究概况 (2)
1.3 榕属分子生物学研究概况 (4)
1.4 榕属植物研究遇到的困难 (5)
2 SSR分子标记研究综述 (10)施丹丽
2.1 DNA分子标记简介 (10)
2.2 SSR分子标记简介 (10)
2.2.1 SSR分子标记的概念 (10)
湖南工业职业技术学院学报2.2.2 SSR分子标记的应用 (12)
3 中国榕属冠毛榕-异叶榕复合的SSR研究 (15)
3.1 实验材料 (15)
3.2 植物DNA提取与检测 (16)
3.2.1实验器材 (16)
3.2.2实验试剂 (16)
3.2.3 DNA的提取 (18)
3.2.4 DNA的检测 (19)
3.3 引物的筛选 (19)
3.3.1 SSR扩增 (20)
3.3.2聚丙烯酰胺凝胶电泳 (21)
3.3.3银染 (23)
3.3.4 引物筛选结果 (24)
3.4 冠毛榕-异叶榕复合的SSR扩增 (25)
3.4.1 冠毛榕-异叶榕复合的SSR扩增方法 (25)
3.4.2冠毛榕-异叶榕复合的SSR扩增结果 (26)
3.5 数据统计与分析 (27)
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