【作者】 李明；

【导师】 高宝嘉；

【作者基本信息】 河北农业大学 ， 森林培育， 2013， 博士

【摘要】 油松是中国特有乡土树种，是北方温带针叶林中主要建群种。了解该物种的适应性和变异特征对油松资源管理和利用具有重要意义。本文以8省10样地天然油松居群及承德人工林为研究对象，从分子、生理、表型三个水平进行研究，借助方差分析、相关性分析、主成分分析等手段，对不同生境差异下天然油松次生林及人工林的DNA水平多态性、油松针叶生理性状、表型性状、矿物质含量等进行分析，探究不同环境胁迫下油松的变异特征和适应机制。主要研究结果如下：确定CTAB法为油松针叶DNA提取的最佳方法；建立了油松ISSR PCR最佳的反应体系（20μL）：2μL10×Buffer、1.0U Taq DNA聚合酶、Mg²⁺2.0mmol/L、模板DNA100μg/20μL、0.20mmol/L dNTPs、0.4μmol/L引物。扩增程序为94℃预变性5min，94℃变性30s，49℃～55℃退火45s，72℃延伸2min，35个循环；72℃再延伸7min；4℃保温。不同地理分布区天然油松居群多态位点比率差异明显；Shannon多样性指数范围为0.192～0.249，平均为0.217。居群间变异占居群总变异的47.55%。聚类分析表明，河南宝天曼、承德大窝铺、宁夏苏峪口和甘肃冶力关居群为一组，辽宁医巫闾山、山西沁源灵空山、陕西蔡家川林场、山西和顺云龙公园和山西汶水三道川林场居群为一组，山东蒙山居群独立为一组。经Mantel检验，油松居群间地理距离和遗传距离不存在显著相关性。温度相关因子（年均温、1月均温、极端最低温）、海拔及年降雨量显著影响天然油松居群DNA水平多态性和生理表型性状。天然油松居群分子变异存在一定的地理变异规律。不同环境因子影响下油松生理、表型等性状表现出响应地理分布区环境的变异。研究结果表明，地理隔离不是油松居群遗传变异的主要原因，温度和水分显著影响天然油松居群DNA水平多态性；群落物种多样性与油松居群DNA水平多态性关联密切。不同海拔天然油松居群DNA水平多态性差异显著。响应海拔变化，1274m～1354m海拔的油松居群DNA水平多态性最高；不同海拔高度的生态因子、低基因流等对油松居群间的遗传分化影响较大。油松针叶脯氨酸和可溶性糖含量在高、低两个海拔区有显著变异。油松针叶木质素含量、纤维素含量变异程度随海拔升高而增加。油松针叶表型性状随着海拔变化均具有显著差异。结果说明，1000m～1354m海拔油松居群DNA水平多态性高；油松针叶生理性状、表型性状受海拔生境影响显著，中海拔区域油松生理活性高，抗逆性强。不同群落类型天然油松居群Shannon信息指数大小排序为白桦油松混交林＞白桦落叶松油松混交林＞油松纯林＞白桦蒙古栎油松混交林；4群落类型油松居群DNA水平多态性间存在明显差异。油松居群DNA水平多态性与油松枝下高及群落辛普森指数呈显著正相关性。响应油松居群土壤矿物质含量增加，油松居群DNA水平多态性具有降低趋势。在不同群落类型间，油松针叶矿物质含量存在显著或极显著性差异。油松针叶比叶面积、干物质含量、叶面积、长/宽等性状在群落类型间存在显著或极显著性差异。说明，油松白桦混交林相对于其他3种群落类型的油松居群DNA水平多态性更高。不同群落类型生境影响油松生理性状和表型性状。不同受害程度人工油松居群DNA水平多态性响应受害程度加深而增加。总遗传变异中，71.04%存在于居群内。响应油松受害程度增加，油松针叶脯氨酸含量减少，受害轻和受害中度油松居群含量相似，差异不显著。受害最重林分叶干物质含量及比叶面积值均最高。说明，不同受害程度油松居群表现出不同的DNA水平多态性，响应受害程度加深，油松居群DNA水平多态性增加，油松抗逆性降低，生理活动受到限制。通过主成分分析，概括了不同生境油松居群及环境因子的主成分。确立了天然油松居群DNA水平多态性高的地理分布区及主要胁迫环境因子；温度、水分相关因子显著影响天然油松居群DNA水平多态性及针叶生理表型性状；确定中海拔高度油松居群DNA水平多态性、针叶理化性状指标最高，是油松生长最佳海拔生境；获得随受害程度加深，油松居群的DNA水平多态性增加变异规律。总之，利用主成分分析，可有效概括油松居群DNA水平多态性、生理性状、表型性状等指标，进而可确定油松居群响应环境差异或胁迫的变异规律和适应性变异。更多还原

【Abstract】 Chinese pine （Pinus tabulaeformis Carr.）, a species endemic to China, is a mainconstructive species of temperate coniferous forest in northern China. It is important tomake clear the adaptability and variation characteristics of the pine for pine resourcemanagement and utilization. In this study, plant survey and sampling were performed inten plots of natural pine populations in eight Provinces and pine plantations in Chengdecity, Hebei province. The genetic polymorphism, physiological traits, coniferousphenotypic traits and mineral contents were analyzed by molecular marker technique ofinter-simple sequence repeat （ISSR）, analysis of variance, correlation analysis inconjunction with principal component analysis. By analyzing the variation characteristicsof Chinese pine in different habitat conditions, the response mechanisms of Chinese pine todifferent environmental stress were investigated. The results are as follows:The results showed that the modified CTAB was suitable for DNA extraction ofChinese pine. A reaction system with volume of20μL contains2μL10×PCR Buffer(Mg²⁺free),1.0U Taq DNA polymerase,2.0mmol·L-1Mg²⁺,100ng template DNA,0.20mmol·L-1dNTPs and0.4μmol·L-1primer. The optimal amplification program was5min ofpre-denaturalization at94℃;35cycles of30s for denaturalization at94℃,45s of anneal at49℃⁵5℃,2min of extension at72℃; and7min of extension at72℃in the final cycleThere was significant differences in polymorphic loci ratio of natural Chinese pinepopulations among different geographic distribution areas. With a range of0.1923～0.2490,the average value of Shannon’s information indexes was0.2165. Analysis of molecularvariance （AMOVA） showed that inter-population variation was47.55%of the totalmolecular variation. Mantel test showed that the correlation of the pine populationsbetween genetic distance and geographic distance was not significant. Temperature factors（annual mean temperature, the average temperature in January, extreme lowesttemperature）, elevation and rainfall significantly influenced the genetic polymorphism andphysiological characters. There were general geographic variation regularities in molecularvariation of the natural Chinese pine populations. In respond to environmental differenceamong different geographical distribution areas, the definite variation of genetic,physiological and phenotypic traits aroused, but the geographic distance was not the main reason of those variations. The result showed that the genetic diversity of Chinese pinepopulations was closely associated with temperature and water conditions, and the geneticpolymorphism were significantly influenced by the species diversity.The genetic polymorphism level of Chinese pine populations at different altitudes hadsignificantly difference. Chinese pine populations at MH altitude （1274m～1354m） hadthe highest level of genetic polymorphism. Ecological factor at different altitude and lowgene flow greatly influenced genetic differentiation. There was a significant variation ofproline and soluble sugar content in the pine’s leaf between low and high altitude areas.Variation of lignin and cellulose contents increased with elevation changes. Phenotypictraits of Chinese pine population were significantly different in response to elevationchanges. Results indicated that Chinese pine populations in the mid-elevation had thehighest genetic polymorphisms level. Physiological and phenotypic traits of naturalChinese pine populations were significantly affected by habitats conditions at differentaltitudes.The order of Shannon’s information index in Chinese pine populations of differentforest community types was as follows: Chinese pine-Bitch mixed forest＞Chinese pine-Larch-Bitch mixed forest＞Chinese pine pure forest＞Chinese pine-Mongolian oak-Bitch mixed forest. Genetic polymorphism of Chinese pine’s populations among thecommunity types had significant differences. Genetic polymorphism of Chinese pinepopulations was significantly and positively correlated with heights under branch ofChinese pine and Simpson indices of the community. The level of genetic polymorphismsdecreased with increasing soil mineral content. The mineral content of Chinese pine leaf indifferent community types had a significant or most significant difference. Coniferoustraits of Chinese pine leaf, including specific leaf area, dry matter content, leaf area,length/width, were significantly or most significantly different between community types.The level of genetic polymorphisms of Chinese pine-birch mixed forest was higher thanthat of other three community types. Thus the physiological traits and phenotypiccharacteristics of Chinese pine leaf were influenced by different population habitat types.Genetic polymorphism level of the artificial Chinese pine populations increased withthe increasing of damage degrees.71.04%of total genetic variation was fromintra-population while28.96%was from inter-population. The higher the damage degrees,the lower the proline content of Chinese pine. There was no significant difference inproline content of Chinese pine between populations with the middle damage degree andpopulations with the most serious damage degree. There were the highest level of leaf drymatter contents （LDMC） and specific leaf areas （SLA） of Chinese pine in population withthe most serious damage degrees. The results indicated that the level of geneticpolymorphism was in response to different damage degrees of Chinese pine population.With increasing of the damage degrees, the levels of genetic polymorphism increased, stress resistance decreased and physiological activity strengthened.Based on principal components analysis, geographic distributions of high levelgenetic polymorphism populations and major stressing factors were identified. Thisresearch showed that temperature and moisture factors significantly influenced geneticpolymorphism and physiological phenotypic traits of Chinese pine. In the mid-altitude, thelevels of genetic polymorphism and the value of physical-chemical indicators of Chinesepine population were the highest. Chinese pine populations adapted best to the growthcondition in mid-altitude. The deeper the degree of damage, the higher the level of geneticpolymorphism of Chinese pine populations. In summary, the genetic polymorphism,physiological traits, phenotypic traits of Chinese pine were effectively summed up on thebasis of principal component analysis, and furthermore to determine variation and adaptivemechanism of Chinese pine populations in response to environmental discrepancies orstresses.更多还原