【作者】 李毳；

【导师】 王孟本；

【作者基本信息】 山西大学 ， 生态学， 2008， 博士

【摘要】 油松(Pinus tabulaeformis Carr.)根系发达,极耐干旱贫瘠,具有良好的保持水土、涵养水源及改良土壤的作用,是我国华北及西北荒山绿化和营造生态林的重要树种。山西是天然油松林分布面积最大的省份之一,也是油松的分布中心,以油松为建群种构成的寒温性针叶林是分布于山西关帝山、太岳山和中条山等地的优势植被类型,在山西森林生态系统中占着举足轻重的地位。然而以往对油松遗传多样性的研究多集中于形态学、细胞学和蛋白质水平。分子水平的研究工作刚刚起步,油松的许多遗传指标仍知之甚少,这对于油松天然林的合理保护和可持续经营是远远不够的。RAPD分子标记和ISSR分子标记是研究种群遗传结构的两个非常有效的工具。本研究在对山西油松地理分布实地考察和全面分析的基础上,选取历山、紫团山、灵空山、芦芽山和关帝山5个天然油松种群作为研究对象。运用RAPD和ISSR标记两种方法,检测天然油松种群的遗传结构及其空间分布格局,探讨各种生态因子与遗传变异的关系,揭示油松对复杂生境的分子适应机制。运用两对引物扩增了叶绿体trnT-trnL和trnS-trnG基因间隔序列,为松属基因组的比较和系统进化研究提供重要信息。主要的研究结果如下:1.山西油松种群的多态位点百分率和多样性指数。用15个RAPD引物对油松种群140个个体进行PCR扩增,共扩增出125个位点,其中多态位点99个,占79.20%;Nei多样性指数和Shannon多样性指数分别为0.2853和0.4232,在5个油松种群中其变化范围分别介于0.2243～0.2626和0.3352～0.3860之间。5个ISSR引物共扩增出35个位点,多态位点28个,占80.00%;Nei多样性指数和Shannon多样性指数分别为0.3085和0.4468。两种标记估算的多样性能比较一致地反映各种群的遗传多样性,即灵空山种群和关帝山种群拥有较高的遗传多样性,芦芽山种群的遗传多样性水平相对较低。通过与松属其它物种的比较,油松种群遗传多样性在松属中处于中等偏高水平。2.山西天然油松种群的遗传结构。RAPD标记扩增的油松种群遗传多样性指数为0.2853,种群内变异为0.2428,遗传分化系数为0.1491;ISSR扩增的油松种群的遗传多样性指数为0.3085,种群内变异为0.2667,遗传分化系数为0.1356。两种标记反映出油松种群间均存在一定程度的变异和分化,在总的遗传变异中85.00%以上的变异存在于种群内,种群间的变异近15.00%。遗传变异主要存在于种群内,种群间的变异虽然不是遗传变异的主要来源,但这是不可忽视的,说明各种群间已经产生一定的遗传分化,从种群分布的自然地理环境分析可知,种群遗传结构格局的形成是环境因素、人为因素和物种本身生物学特性综合作用的结果。3.5个油松种群间的遗传关系。基于ISSR标记,根据Nei的方法计算油松种群的遗传相似系数和遗传距离。芦芽山种群和关帝山种群遗传一致度最高为0.9716,遗传距离最小为0.0288,这两个种群的相似程度最大;紫团山种群和关帝山种群遗传一致度最低为0.8999,遗传距离最大为0.1054,这两个种群亲缘关系相对较远。RAPD标记和ISSR标记反映5个种群间亲缘关系的远近稍有不同,但UPGMA的聚类结果完全一致。灵空山种群和紫团山种群首先聚在一起,再与历山种群聚为一组,芦芽山种群和关帝山种群聚为另一组,2组再聚在一起,地理距离相近的种群首先聚在一起。4.油松叶绿体DNA间隔序列的特点及应用。由于叶绿体DNA的编码区和非编码区分子进化速度不同,因而适用于不同阶元的系统学研究。本研究用两对特异引物扩增油松叶绿体非编码区trnT-trnL和trnS-trnG间隔序列,序列对比结果显示这两个片段在物种水平十分保守和稳定,而在松科各属间变异位点丰富,说明这两个序列片段不适合作为物种以下种群间的分析,适合作为物种以上的分子标记和系统学研究。结合GenBank(http://www.ncbi.nlm.nih.gov)中已有的序列资料,下载其它有关的松科植物trnT-trnL和trnS-trnG序列,一起进行分支分析,构建邻接系统进化树。分析了油松与其近缘物种的亲缘关系,为松科属间或种间植物提供更多更深入的信息。山西高原油松种群遗传多态性主要来自等位基因频率的不同,而不是等位基因的组成的差异。说明部分个体的死亡不会发生基因丢失,但个体数的减少会使种群在某些位点有遗传漂变的趋势,降低种群适应环境的能力,从而影响到种群的质量和进化潜力。对油松天然林保护要有科学的理念,长远的策略,保护生境,减少人为干扰,使其不片断化是油松资源保护工作的重点。更多还原

【Abstract】 Chinese pine,Pinus tabulaeformis Carr.has a broad distribution across northeastern to southwestern China.It has a strong adaptability to dry and barren habitat,meanwhile,it is a fast-growing tree species and possesses a strong ability to develop roots.Therefore it also has the function of conserving soil and water,enriching headwaters and improving soil fertility.The species therefore can be a main species for afforestation in the semiarid and waste mountains in northeast of China.Shanxi Plateau has long been known as its distribution center,where the tree predominate the forest landscape.However,natural Chinese pine forests have long been affected by natural processes and anthropogenic perturbation without systematic management,leading to serious ecological hazards and partial loss of ecological functions.Understanding of the genetic diversity and genetic structure of species is necessary for the conservation of species and populations,especially the conifer species.Many studies on the levels of morphology,chromosome,isozyme from Chinese pine have been conducted in recent decades.However,the genetic diversity and structure of the Chinese pine populations on the genomic level remains unknown.Little is known about how genetic variation is distributed within and among populations,despite the species considerable ecological importance.On the base of analysis of geographical distribution of Chinese pine and of the field investigation,the Lishan in Zhongtiao Mountains,Zituan Shan in Taihang Mountains,Lingkong Shan in Taiyue Mountains,Luya Shan in Guanqin Mountains,and Guandi Shan in Lvliang Mountains were selected as investigated fields,which represent the natural distribution of Chinese pine in Shanxi Loess Plateau.Neutral molecular markers such as random amplified polymorphic DNA(RAPD) and inter simple sequence repeats(ISSR) are well established in population genetics and have proven to be useful tools for the detection of plant population variability and differentiation.In our study,both ISSR and RAPD markers have been used to survey the genetic structure at different levels in P.tabulaeformis(within and among populations) and the correlations between genetic variation and environmental system and the mechanism of ecological fitness and molecular evolution.Two pairs of primers were used to amplify noncoding region of chloroplast trnS-trnG and trnT-trnL from genome of chloroplast,respectively.The resultant sequences were analyzed by alignment and the phylogram trees were constructed using a series of softwares to inference the systematics relationship of the Pinaceae and Pinus,and to provide the significant information for the comparison of genome and systematic evolution of Pinus.The findings of this research are as follows:RATIO OF POLYMORPHIC LOCI AND INDEX OF DIVERSITYA total of 125 reproducible bands were obtained from 140 individuals of P. tabulaeformis using 15 RAPD primers.Ninety-nine polymorphic loci were identified from 125 reproducible RAPD bands(79.2%).Nei’s analysis of genetic diversity ad Shannon’s information index are 0.2853 and 0.4232, respectively,which varies in five populations of P.tabulaeformis range from 0.2243～0.2626 and 0.3352～0.3860,respectively.A total of 35 reproducible bands were obtained from 140 individuals by using 5 ISSR primers. Twenty-eight polymorphic loci(80%) were identified.Nei’s analysis of genetic diversity and Shannon’s information index are 0.3085 and 0.4468,respectively. The genetic diversity of five populations was compared and ranked by using the diversity index measured by two markers.The results indicated that the taxis of diversity of five populations base on RAPD and ISSR were similar except for the LS population.The genetic diversity of P.tabulaeformis is high on Lingkong Mountain and Guandi Mountain and low on Guanqin Mountain.The genetic diversity of P.tabulaeformis is relatively in the middle to high level compared with that of species in Pinus.THE GENETIC VARIATIONS AND DIFFERENTIATIONSThe majority of genetic variations were found within populations and some genetic differentiation occurred among populations of P.tabulaeformis.Base on the RAPD marker,the total genetic diversity(Ht) and genetic diversity within populations(Hs) of P.tabulaeformis are each 0.2583 and 0.2428,and the corresponding differentiation among populations(Gst) are 0.1491.Base on ISSR marker,the Ht and Hs of P.tabulaeformis are 0.3805 and 0.2667, respectively,and Gst is 0.1356.The data from two molecular markers suggest the existence of genetic variation and differentiation in population of P. tabulaeformis in Shanxi.The majority variation(85%) was found within populations.Though genetic variation among population(15%) is not large,it should not be negligible.Base on the analysis combining with natural geographic environmental factors,we concluded that the effects on the development of pattern of genetic structure of P.tabulaeformis population involved geographic location,natural habitat and human perturbation.GENETIC IDENTITY AND GENETIC DISTANCE AMONG POPULATIONS OF P.TABULAEFORMISThe genetic identity and genetic distance among five P.tabulaeformis populations were calculated using Nei’s original measure.The largest genetic identity(0.9716) and the smallest genetic distance(0.0288) were found between LY population in Guanqin Mountains and XW population in Guandi Mountains, and similarity of two populations is highest.Meanwhile,the least genetic identity(0.8999) and biggest differentiation(0.1054) were found between ZT population in Taihang Mountains and XW population in Guandi Mountains.The UPGMA dendrogram tree was constructed by using genetic distance of five populations to further confirm the genetic relationship among populations in Shanxi.THE PHYLOGENETIC RELATIONSHIP OF PINUS INFERRED FROM CHLOROPLAST DNAChloroplast DNA(cpDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels corresponding to different development rate of the encoding and nonencoding regions in genome of chloroplast.In this study,two pairs of specific primers were used to amplify noncoding region of chloroplast trnS-trnG and trnT-trnL from chloroplast genome,respectively.Alignment of the sequences of P.tabulaeformis from different population indicates that these two DNA sequences are identical and conserved among five populations even within genus and varies among genera of Pinaceae,suggesting the two DNA sequences are not fit to being used to analyses the systematic relationship within species and could be used for that among species within familia.In this study,we constructed the phylogenic tree by combining the chloroplast trnS-trnG and trnT-trnL sequences amplified from five P.tabulaeformis populations and downloaded from GenBank (http://www.ncbi.nlm.nih.gov).The phylogenic relationship between P. tabulaeformis and its relative species were analyzed combining with the morphology,which will provide much more information for elucidating the genetic relationship among species and genera of Pinaceae.CONSERVATION OF PINE FORESTThe genetic diversity of pine populations in Shanxi plateau mainly rests on the different allelic frequency rather than on the composition of alleles,indicating that the disappearance of some individuals does not lead to a loss of genes. However,a decrease in individuals may cause genetic drift and inbreeding leading to a decline in fitness with environmental or disease-related reductions in productivity for Chinese pine populations.Furthermore it will affect the evolution potential of the pine population.We should protect the natural pine forest with scientific consciousness.The conservation of pine forest should be focus on the protection of environment and decrease the artificial perturbation, which may cause the fragments level of the pine forest.更多还原