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摘 要
棕榈科是泛热带分布的科，棕榈（Trachycarpus fortunei）是其中少数可以分布到温带的植物，在中国的分布遍及长江以南各个省区。本文通过对棕榈标本和文献的查阅，结合对云南、广西、湖南三省棕榈的实地调查和观察，分析棕榈的分布范围和居群特征。棕榈居群内个体数少，幼树和幼苗极少，年龄结构不合理。同时对棕榈属植物的分布进行分析。
从棕榈的干燥叶片或新鲜叶片中采用CTAB法摸索和提取棕榈基因组DNA，找出最好的提取条件和药剂用量。在此基础上，对棕榈ISSR－PCR进行扩增，对影响ISSR反应的各因子（模板DNA浓度、Mg2+用量、dNTP浓度、退火温度、热循环数及不同厂家生产的药品与仪器）进行探讨，确定了该研究的最佳反应条件，建立了棕榈ISSR反应的最佳反应体系，为进行棕榈居群遗传多样性的研究奠定了基础。摸索获得的最佳ISSR反应条件应用于棕榈13个居群200个样本的扩增。从100条ISSR引物中筛选出可以扩增出清晰、重复性好的条带的引物，结果共产生了107条带，其中102条具有多态性，扩增条纹的大小在300－1900bp,多态性百分率为95.33％，单态率为4.67％，平均每个引物能扩增出8.9条带。棕榈的遗传多样性及分化系数由POPGENE软件获得。棕榈物种水平的遗传多样性（Ht）为0.3234，居群内遗传多样性(HS)是0.1982。棕榈的(Gst)值为0.3827，即38.27%的变异存在于居群间，61.73%的变异存在于居群内，棕榈的遗传变异主要存在于居群内。Nei 遗传多样性指数h为0.3218，Shannon’s信息指数I为0.4843。
由UPGMA法获得的基于Nei的无加权计算遗传距离棕榈各居群的聚类图。棕榈各居群的遗传距离在0.06和0.27之间， 遗传距离最小的是0.06在广西黄沙镇居群GHS6和广西花坪自然保护区居群GHP7之间，其次是DWS1和DWS2, DWS2 和 DWS3，DYX4和DFG5之间，遗传距离为0.09。遗传距离最大的是0.27在DWS1和GHS6, GHS6 和 XAR10之间，次大的为0.26在D1和GHP7之间。这13个居群的棕榈分为4组，第一组为居群DWS1, DWS2和DWS3；第二组为居群DYX4, DFG5, GHS6和GHP7；第三组为居群XAR10, XYY11, XZJ12和YFL13；第四组为居群GJX8和GNOP9。从系统图上可以看出来自云南省文山县的三个居群（DWS1, DWS2和DWS3）有着更近的亲缘关系而聚在一起；居群GHS6和GHP7，居群XAR10, XYY11和XZJ12 在遗传上都有很近的亲缘关系，这些与它们的地理距离的大小是统一的。然而也有部分居群的遗传关系与地理距离不相符。
各居群的遗传多样性从高到低依次是：XAR10 > DYX4 > GHS6 > DWS3 > DFG5 > DWS2 > XYY11 > GNP9 ＝ XZJ12 > GHP7 >YFL13 > DWS1 > GJX8。遗传多态性最高的居群是湖南安仁的XAR10居群，其次是云南玉溪DYX 4居群，多态性比率最低的是广西的GJX8。
棕榈居群内存在巨大的遗传变异，适应力强。丰富的遗传多样性为棕榈提供了变异的源泉。棕榈分布广，栽培历史悠久，但野生居群都比较小，多呈岛屿状分布格局，提出了棕榈的保护策略。
关键词：棕榈,遗传多样性,ISSR标记,居群,棕榈属
Abstract
Palm plant is considered to be a symbol of tropical or subtropical landscape, Trachycarpus fortunei is a species with widespread range in south of River Changjiang. Based on the field investigation, note of speciman and literatures, the distribution and of populations T. fortunei were probed. T. fortunei distributed mainly in Yunnan, Guangxi, Guangdong, Hunan, Hubei, Zhejiang, Sichuan, Fujian, Guizhou, Shanxi, Jiangxi and Gansu.The popultion size of T. fortunei are small, number of young tree small and the age struction inconsequence. The distribution of genus Trachycarpus were analysised and its origin been discussed.
Genomic DNA was isolated from leaf samples using the improved hexadecyl- Cetyl Trimethyl Ammonium Bromide (CTAB) method. Inter-Simple Sequence Repeat (ISSR) is a good molecular marker for revealing genetic diversity. Reaction system differed with difference species, so optimization of ISSR-PCR reaction is very important. The study discussed mainly the factors impacting the banding patterns of PCR. To optimize the reactions, several parameters were tested, including the concentration of DNA template, MgCl2, dNTP, primer and Taqpolymerase with different annealing temperature.
In this study Inter-simple sequence repeat (ISSR) method revealed genetic diversity of 200 individuals among 12 natural populations from three province Yunnan, Guangxi and Hunan and one cultivated population from Shenzhen FairyLake Botany Garden. Twelve of 100 ISSR primers can amplify clear bands, and produced 107 loci, 102 of which is polymorphic, the percentage of polymorphic loci is 95.33%. The band size ranged from 300 to 1900bp, with an average of 8.9 bands per primer with an average of 8.5 bands per primer. The indices of genetic diversity and gene variation were computed by the POPGENE in this study. The value of total gene diversity (HT) is 0.3234, gene diversity within population (HS) is 0.1982. The value of (GST) is 0.3827. The gene variation remains mainly within population. Nei’s gene diversity h = 0.3218, Shannon’s information index I = 0.4843.
The dendrogram based on Nei''s genetic distance by the UPGMA method. The genetic distance between populations is between 0.06 and 0.27, the shortest genetic distance is 0.06 between GHS6 and GHP7. The second shortest genetic distance is 0.09 between DWS1 and DWS2, between DWS2 and DWS3 and between DYX4 and DFG5. The longest genetic distance is 0.27 between DWS1 and GHS6, between DHS6 and XAR10. The second longest genetic distance is 0.26 between DWS1 and GHP7.
The 13 populations formed four groups according to the dendrogram. The first group is population DYX4, DFG5, GHS6 and GHP7; the second group is population XAR10, XYY11, XZJ12 and YFL13; the third group is GJX8 and GNOP9; the forth group is population DWS1, DWS2 and DWS3. Populations in Wenshan (DWS1, DWS2 and DWS3) showed a closer genetic relationship, Population GHS6 and GHP7, XAR10 and XYY11 and XZJ12 have a closer genetic relationship. Their genetic distance accord with geographic range. The cultivated population YFL13 and population XZJ12 has a closer genetic relationship.
The rank of the percentages of polymorphic sites of 13 populations was: XAR10 > DYX4 > GHS6 > DWS3 > DFG5 > DWS2 > XYY11 > GNP9＝XZJ12 > GHP7 > YFL13 > DWS1 > GJX8. The highest population of gene diversity is Hunan Anren population XAR10, the second is population Yunnan Yuxi DYX4. The lowest genetic diversity is Guangxi Jinxiu population GJX8.
The genetic polymorphic of T. fortunei is very high, and mainly variations are within populations. T. fortunei is a widespread species, has been used for thousand years. Its population size is small and geographic range is wide.
Key Words: Trachycarpus fortunei; Genetic diversity; ISSR marker; Population; Genus Trachycarpus
目录：
摘 要II
AbstractIV
1.引 言2
1.1棕榈属植物的地理分布2
1.1.1 棕榈属种的主要形态特征及命名2
1.1.2 棕榈属的地理分布6
1.1.2.1 棕榈7
1.1.2.2对叶棕榈7
1.1.2.3 宽裂棕榈7
1.1.2.4 山棕榈7
1.1.2.5龙棕7
1.1.2.6 泰国棕榈8
1.1.2.7 石门棕8
1.1.2.8 喜马拉雅棕榈8
1.1.2.9 魏格纳棕榈8
1.1.3 棕榈属的利用情况8
1.1.3.1棕榈（T. fortunei）广泛的应用9
1.1.3.2 棕榈属其它种的应用9
1.3 分子标记的选择及在棕榈科居群遗传多样性中的应用10
2. 材料与方法14
2.1 材料14
2.1.1野外调查与植物材料采集14
2.1.2 主要化学试剂15
2.2 研究方法16
2.2.1 DNA提取16
2.2.1.1 所用药品的配制16
2.2.1.2 DNA提取过程16
2.2.2 PCR扩增17
2.2.2.1 预扩增17
2.2.2.2 对影响ISSR－PCR扩增效果的各成分与条件设置梯度18
2.2.3利用琼脂糖电泳对DNA与扩增产物进行检测19
2.2.3.1 DNA的检测与定量20
2.2.3.2 扩增产物的检测20
2.2.4 数据分析及主要遗传参数21
3.结果与分析23
3.1 棕榈在中国的地理分布23
3.2 ISSR-PCR反应条件筛选与优化26
3.2.1模板DNA浓度对ISSR反应体系的影响26
3.2.2 Mg2+对棕榈ISSR的影响26
3.2.3 dNTP浓度对ISSR反应的影响27
3.2.4 引物浓度对ISSR反应的影响27
3.2.5退火温度及热循环数对ISSR的影响27
3.2.6 TaqDNA聚合酶与PCR厂家及型号的不同对ISSR扩增效果的影响27
3.2.7 反应条件的确定27
3.3 由ISSR揭示的棕榈的遗传多样性28
3.3.1 棕榈物种水平的遗传多样性28
3.3.2 棕榈各居群的遗传多样性比较29
3.3.3 棕榈的遗传结构31
3.3.4 各棕榈居群的聚类分析33
4. 讨 论36
4.1 棕榈属植物的分布格局与起源初探36
4.2关于分子标记ISSR36
4.3 ISSR-PCR反应条件筛选与优化37
4.4 棕榈居群的遗传结构39
4.5 棕榈的保护40
主要参考文献42
附 录：49
附表1 缩写词及英汉对照49
附表2 实验中所用ISSR引物50
附图1 引物810对居群XAR的扩增51
附图2 引物810对居群GJX8的扩增51
附图3引物864对云南文山居群DWS1, DWS2, DWS3的扩增51
致 谢52
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