侧柏种源遗传多样性与地理变异规律研究.pdf
侧柏种源遗传多样性与地理变异规律研究 Genetic Diversity and Geographic Variation of Platycladus orientalis (L.) Franco Provenances Abstract Orientalis (Platycladus orientalis (L.) Franco) originates in China.Its germplasm resource is very abundant. Orientalis is an important tree species of afforestation on the rocky barren.According to the reality of the lack of good provenances. The diversity of four orientalis intraspecific populations was analyzed using morphological traits, the genetic diversity and population genetic structure for four geo-ecological groups using principles of molecular systematic and fluorescent-AFLP marker techniques were studied by morphology traits and molecular markers, respectively, in order to provide science evidence for conservation and utilization, to select good provenance and to divid provenance region and to allocate seed The main results are as follows: 1. The diversity of growth traits of 41 provenances and cones of 21 provenances in 17 provinance of P.orientalis were investigated. The results indicated: There were significant differences among and within provenances. The growth traits have great diversity with the variation coefficient (CV), the range varied from 5.41% to 47.55%. The variation coefficient of branching angle was highest (28.74%), and the one of tree height was lowest; The variation coefficient of seeds was from 4.77% to 47.42%, and the variation coefficient of the seed thickness was the highest, the one of the seed length was the lowset. The variation coefficient of the seed traits is lower than the growth traits, this showed that the genetic stability of seeds is higher.There are siginificantly diversity of the variation coefficient and the relative range in the cone shape and the growth traits, the analysis indicated that there are abundant genetic diversity in the P.orientalis. 2. The largest morphological differentiation coefficient of the growth traits among the orientalis was the tree height（Vst= 46.82%）, the smallest was the crown（Vst = 20.92%）. The average morphological differentiation coefficient of the growth traits was 30.12%, in means that the Vst within the provenance was 69.88%;The range of the morphological differentiation coefficient was from 16.78 to 27.66. The average of the Vst is 22.92%, the genetic variation among provenance accounted for 77.08% of total variations. This suggested that the variation of morphological traits among provenance of orientalis is much higher than within provenance. 3. The result of grey correlative analysis between the growth traits, the seed traits and 4 山东农业大学博士学位论文 the latitude, longitude indecates: The correlation of between all traits and the latitude was greater than the longitude, this showed that the morphological diversity of the orientalis mainly came from the latitudinal variation. The typical correlation analysis between the 44 provenances with 8 different traits of the cone showed: the geography variation of oreitalis was a variation that latitude mainly brought on. Its variation mode was two-way variation from latitude and longitude. 4.Spatial correlation analysis showed that global morans I of growth characters is from -0.1878 to 0.1921,the global morans I of seed is from -0.4842 to 0.7518, most autocorrelation coefficient is not significant,the result showed that presents certain correlation over short distance, while presented spatial uncorrelation or spatial random with distance increases. Global morans I of DBH is highter than tree height,indicated that variation of DBH is highter than tree height.Local G coefficient of growth characters is from-1.097 to 5.6426,while the seeds is from -2.1899 to 1.9142,and most of them is negative, showed a lack of spatial structure,around provenance is smaller. Local G coefficient of few of northern and southern provenance is 0,they are randomly distributed. 5. The semivariance analysis showed that the nugget of DBH growth and height growth were respectively 58.72% and 47.78%, both of which had moderate spatial correlation. While the nugget of branch angle was as high as 90.6%，and the spatial correlation was very weak. When analysis in krige method were performed, the growth Characters of the provenances of platycladus orientalis had certain kinds of spatial structures which presenting a non-random distribution such as progressive change，bi-directional progressive change and irregular patch. Analysis among the provenances P. orientalis also showed a complex pattern that most growth characters seldom have any spatial structure，while A small proportion of the growth characters have spatial structure any way. 6. The heritability in, behaved differently between the Pingyin and Zaozhuang sites. At Pingyin site, the heritability in height and DBH was respectively 0.70490.9557 and 0.70070.8858. At this site, the tree height had higher heritabilities. At Zaozhuang site, the heritability in height and DBH was respectively 0.26840.8102 and 0.32620.9424. At this site, the DBH had higher heritabilities than the tree height had. Totally, trees in 23 years old had lower heritabilities of both in DBH and tree heigh than young trees had. The correlation analysis between geographical coordinate and annual increment of trees in different forest ages in Platycladus orientalis had the following results: 5 侧柏种源遗传多样性与地理变异规律研究 The correlation coefficient between tree height and longitude tended to become weaker with forest got older, while the correlation coefficient between tree height and latitude tended to become stronger with forest got older. Correspondingly, the correlation coefficient between tree DBH and longitude tended to become stronger with forest got older, while the correlation coefficient between DBH and latitude tended to become weaker with forest got older. 7Population genetic structure was studied using fluorescent-AFLP markers on 256 orientalis (P. orientalis (L.) Fr.) accessions collected from the Southern group, the Northern group, the Central of Chian group and the Shandong province group of fluorescent-AFLP markers. The purpose of this study was to determine the genetic structure and genotypic diversity amongst the different provenance of orientalis. The results showed that the average number of polymorphic loci (A) was 100.4, the percentage of polymorphic loci (P) was 46.47% by 8 pairs of EcoR I /MseI (Mse I - a FAM fluorescent marked primer) primers in orientalis of four groups. Analysis for the average number of polymorphic loci (A) and the percentage of polymorphic loci (P) in four orientalis groups indicated that the Sourthern group (A = 102.1，P = 47.28%) the Northern group (A = 104.2，P = 48.25%) the Cemtral Chian group (A = 102.1，P = 47.28%) the Shandong Province group （A =95.4， P = 44.14%）; The analysis of diversity and genetic structure from four geo-ecological groups showed that genetic diversity of orientalis provenance in the Southern Group is most abundant, then it is the Northern group; the Northern group and the Shandong Provence group were lowest. 8. There was significantly different of genetic diversity among orientalis provenance, the range of is from 1.2622 to 1.6122, is from 1.0983 to 1.2519, the Shannon Index is from 0.1025 to 0.2371. The observed number of alleles (Na), the effective number of alleles (Ne), the Neis gene diversity (H) and Shannon information index (I) at species level were respectively 1.5311, 1.2022, 0.1305 and 0.2102. The observed number of alleles (Na), the effective number of alleles (Ne), the Neis gene diversity (H) and Shannon information index (I) at provenances level were respectively 2.000, 1.2406, 0.1605 and 0.2695. Four parameters at species level was high significant higher than at provenances level. At regional level, Neis gene diversity and Shannon information index (H=0.1434、I = 0.2366） in the Southern group was higher than those in the Northern group （H = 0.1425、 I = 0.2305）and in the Central China group（H = 0.1377、I = 0.2283）, there were no significant differences; but they were significant higer than those in the Shandong Province group（H = 0.1285、 I = 0.2227） . The analysis of four population diversity and genetic structure from four 6 山东农业大学博士学位论文 provenances showed that genetic diversity of orientalis provenances in the Southern Group was most abundant, there were many heterozygous in the Southern group and the Northern group, the genetic diversity of orientalis provenances in the Shandong Province Group was lower. 9. The parameters-genetic differentiation coefficient and gene flow of population genetic structure in orietnalis were analyzed in this study. The genetic diversity at the species level (Ht) was 0.1106, the genetic differentiation coefficient (Gst) among the provenances was 0.2703, This showed that orientalis genetic variation was mainly within the provenances and accounted for 72.97% of total variations, the genetic variation between provenances accounted for 27.03% of total variations. The results from AMOVA analysis showed that the orientalis genetic variation was mainly within the provenances and accounted for 74.86%. The genetic variation of within regional accounted for 11.12%. There was a high level of genetic differentiation at provenances level, and the genetic diversity was mainly at provenances level. 10. The gene flow (Nm) was 1.3804 according to the genetic differentiation coefficient between groups (Nm = 1. 3804). This indicated that there are partly gene exchanges among four regional of orientalis. The gene flow (Nm) at regional level was high significant higher than at species level. Analysis for gene flow (Nm) in four orgional groups indicated that the Northern group (Nm = 3.8442) the Central China group (Nm = 3.3679) the Southern group (Nm = 2.5390) the Shandong province group（Nm = 2.1078）. This showed that the gene exchange was frequently within the small scale region. 11. Neis genetic identities in 26 orientalis provenances were between 0.8422-0.9919, the average was 0.9380; Genetic distances were between 0.00860.1717, the average was 0.0647. It was suggested that there were higher similarity between various geo-ecological groups in orientalis and lower genetic distance. 12. The results from UPGMA cluster analysis for 26 provenances showed that all the orientalis could be divided into 5 groups. Almost all provenances from the same population could be clustered into the same group. Three provenances in groupwhich was call the Southern population came from Ynnan Wenshan, Fujian Nanping, Guizhou Guiyang ; There were 4 provenances found from Northern population in group , which came from Beijing Miyun, Neimenggu Wulashan, Liaoning Chaoyang, and Xinjiang Yili; Totally 3 provenances were clustered in group , Among them, 1 originatel from Sichuan Xichang, 1 from Hubei Jingmen, and the last one is from Henan Qunshan; The group came from 7 7 侧柏种源遗传多样性与地理变异规律研究 provinces of the Central Chian, including Gansu Weixian, Jiangsu Tongshan, Shanxi Huangling, Shanxi Shilou, Ningxia Yinchuan, Hebei Yixian, Zaozhuang and Junan of Shandong Province; The group came from Shandong Province, including 8 provenances, they are Feixian provenance, Qufu provenance, Taian provenance, Boshan provenance, Anqiu provenance, Pingdu provenance, Pingyin provenance and Liaochen provenance. The Mantel test showed: There was a positive correlation between geographical distance and genetic distance (r=0.288 6，P = 0.093 0). The results from UPGMA cluster analysis for four region groups showed that the Southern group and the Central China group was clustered together firstly and the Northern group were clustered together, at last the Shandong Province group were clustered together. This indicated further that the similarity between the Southern group and the Central China group was highest and genetic relationship was closest. Key words：Platycladus oriental s provenances;Morphological characters; Amplified fragment length polymorphism; Genetic structure; Geographic variation 8 符号说明 A：the number of polymorphic loci，多态位点数 AFLP ：amplified fragment length polymorphism，扩增片段长度多态性 CTAB：cetyltriethylammonnium Bromide，十六烷基三乙基溴化铵 DBH：diameter at breast height，胸径 EB：Ethidium bromide，溴化乙锭 Gst：the coefficient of gene differentiation，遗传分化系数 H：Nei's gene diversity，Nei's 基因多样度 h2：Heritability，遗传力 I：Shannon Index，香农信息指数 ISSR：Inter-simple sequence repeat，简单序列重复区间 Moran's I：Moran's I Index，Moran's I 指数 MS：mean square，均方 Na：Observed number of alleles，观测等位基因数 Ne：Effective number of alleles，有效等位基因数 Nm：Gene flow，基因流 P ：P value，P 值 PAGE：Polyacrylamide gel electrophoresis，聚丙烯酰胺凝胶电泳 PCR：Polymerase chain reaction，聚合酶链反应 PPA：The percentage of polymorphic loci，多态位点百分率 PPB：Percentage of polymorphic bands，多态带百分率 RAPD：Randomly amplified polymorphism DNA，随机扩增多态性 DNA RFLP：Restriction fragment length polymorphism，限制性片段长度多态性 i R：Relative range，相对极差 SSR：Simple sequence repeats，简单序列重复 UPGMA：Unweighted pair group method with arithmetic average，非加权算术平均聚类法 Vst：Differentiation coefficient of phenotypic traits，表型分化系数 关于学位论文原创性和使用授权的声明 本人所呈交的学位论文，是在导师指导下，独立进行科学研究所取 得的成果。对在论文研究期间给予指导、帮助和做出重要贡献的个人或 集体，均在文中明确说明。本声明的法律责任由本人承担。 本人完全了解山东农业大学有关保留和使用学位论文的规定，同意 学校保留和按要求向国家有关部门或机构送交论文纸质本和电子版，允 许论文被查阅和借阅。本人授权山东农业大学可以将本学位论文的全部 或部分内容编入有关数据库进行检索，可以采用影印、缩印或其他复制 手段保存论文和汇编本学位论文，同时授权中国科学技术信息研究所将 本学位论文收录到中国学位论文全文数据库 ，并向社会公众提供信 息服务。 保密论文在解密后应遵守此规定。 论文作者签名： 导 师 签 名： 日 期： 山东农业大学博士学位论文 1 引言 1.1 研究意义 近几百年来，由于人口膨胀和经济发展所带来的压力，地球上的生物多样性正在 急剧下降。生态系统正受到严重的破坏，大量物种已经灭绝或处于濒危状态，许多物种 的居群规模正在锐减，种内遗传多样性急剧丧失（葛颂等，1994；Meffe et al.，1994； Avise et al.，1996） 。与此同时，人类对生物资源的依赖和需求却在日益增大，在这些严 酷的事实面前，人们终于认识到，保护物种及其所蕴藏的遗传多样性就是保护生物多样 性， 就是保护人类赖以生存的资源， 就是保护人类自己 （陈灵芝等， 1993； Meffe et al.， 1994） 。因此，开展植物资源的遗传多样性的调查研究，已引起普遍重视。遗传多样性 作为生物多样性的重要组成部分，是生物多样性的基础(葛颂，1998b；顾万春，1998)。 广义地讲，遗传多样性就是生物所携带遗传信息的总和；狭义上，则指种内不同群体和 个体间的遗传多态性的程度，或称遗传变异（施立明等，1993） 。物种的遗传多样性 (Genetic diversity)是其长期进化过程的遗传变异和对环境适应性的产物，是物种可持续 生存和发展的重要基础（季维智，1999） 。遗传多样性研究，一方面，可以帮助我们了 解种质资源多样性状况，了解群体的遗传背景、遗传结构及品种间的亲缘关系，为种质 资源的利用与开发提供帮助，为育种材料的选择提供信息；另一方面，还可以帮助我们 对种质资源进行区划，进而制定合理开发利用的方案和育种策略，为不同地域或生态环 境间的相互引种或驯化提供指导。可见，遗传多样性的研究不仅具有重要的理论意义， 而且具有重要的实践意义（王红霞，2006） 。无论是从保护遗传资源特有基因，还是保 存满足未来需求基因的角度，遗传多样性的保护都是一项紧迫的工作，具有十分重要的 经济、科学和历史文化意义。 侧柏(Platycladus orientalis (L. ) Franco)为柏科常绿乔木植物，别名扁柏、香柏、片 柏、片松（王婷等，2006） ，因其耐干旱瘠薄、抗病虫、寿命长、适应性强，是我国华 北石质山地、 西北黄土高原以及华东、 华中部分地区瘠薄山地造林的重要造林绿化树种。 多年实践证明，侧柏以其适生范围广、保水能力强、绿化美化效果好等特点，显示出它 巨大的绿化潜力（曲绪奎，1987；王彩梅，2004） 。柏树全身都是宝，侧柏种子、材、 叶、皮、根均为人民生活和工业的要原料。柏树木材颜色美丽，纹理细致，材质坚韧、 平滑光泽，含树脂和芳香油，防蛀耐朽，经久不变，是房屋建筑、桥梁、家具、农具、 车船、细木工、雕刻、文具等的上等用材。川、黔、鄂等省的木船制造业柏木是首选优 9 侧柏种源遗传多样性与地理变异规律研究 质用材。 一直沿袭至今， 素有“柏木船”之美称。 柏木的锯末和锯屑干馏后可获得香柏油， 是香烟工业的原料，其剩余物制成卫生香、驱蚊香等，出口到东南亚诸国，备受欢迎。 侧柏叶、种仁、根皮在中药领域占重要席位，侧柏叶气清香，味苦涩、微辛，具有凉血 止血、清肺止咳功效，在临床上常用于治疗出血症、风湿痹痛、高血压、咳喘等（梁统 等，2003；回瑞华等，2005） ；侧柏叶挥发物不仅能有效抑菌抗菌，而且具有抗肿瘤的 明显保健功能（李晓储等，2005） ，另外，侧柏叶的挥发油中 -雪松醇是一种良好的定 香剂、香料（黄致喜等，1999） 。柏子仁含油脂丰富，又含有皂贰及挥发油，性味甘、 平、无毒，人心、肝、肾、大肠经，是著名的滋养强壮药，具有养心安神、润肠通便、 滋肾、祛痰止咳等功效。对于阴血不足所致的心悸、失眠、健忘、神志恍惚和老年性便 秘等症均有疗效。可见，侧柏具有重要的生态、经济和社会效益。 侧柏起源于中国，分布远及日本、朝鲜
