【作者】 陈志丹；

【导师】 孙威江；

【作者基本信息】 福建农林大学 ， 茶学， 2013， 博士

【摘要】 铁观音（Camellia sinensis cv.Tie-guanyin）是原产于福建安溪的国家级茶树良种，性状优异，在茶树育种领域也是优良的育种原始材料。近年来铁观音茶树在栽培中表现出一些性状分化现象，特别是在表型形状上表现出差异性，影响了铁观音茶树种性纯度和经济性状的维持。本研究分析了铁观音茶树的形态学变异性，基于ISSR分子标记技术分析了不同表型性状的铁观音茶树的差异，构建了铁观音茶树的分子指纹图谱，应用cDNA—AFLP技术对两份具有表型典型差异性的铁观音茶树的基因表达差异进行了研究，并对比了部分差异表达的基因在两份铁观音茶树样本上的相对表达量差异，从转录水平探讨了铁观音茶树的性状分化，为鉴别和保护纯种铁观音茶树品种以及研究分析铁观音茶树的性状分化提供参考。主要研究结果如下：1、铁观音茶树表型性状变异性分析调查了80株铁观音茶树的部分形态学特征，对树姿、芽叶颜色、叶缘锯齿数、侧脉对数、叶片厚度、叶片长度和叶片宽度7项表型性状的变异性进行分析，结果表明：铁观音茶树树姿的变异最大，变异系数为48.95%，其次为芽叶颜色，变异系数为36.83%。通过主成分分析提取了3个可作为判别铁观音茶树表型性状分化性参考指标的主成分。遗传相似性分析和聚类分析也显示种性纯度较高的铁观音茶树样本，具有较高的遗传相似性，并且被聚在一个类群中。2、不同表型性状铁观音茶树的分子标记差异优化建立了ISSR扩增反应体系，利用ISSR技术分析了包括不同表型铁观音茶树在内13份茶树样本的遗传多样性和亲缘关系。结果表明：11条引物在13份茶树样本中共扩增出91条谱带，其中45条为多态性条带，多态性比率为49.45%。13份茶树样品的其Nei基因多样性指数为0.17，Shannon信息指数为0.25，表明供试茶树的遗传多样性水平较低。遗传相似性分析表明13份茶树样本的遗传相似系数为0.687-0.986，平均为0.805，遗传亲缘关系较近。聚类分析将供试茶树分为3大类群，9份供试铁观音茶树被聚在同一类群中，其中两份种性纯度较高的铁观音茶树被聚在同一亚类中，两份表型性状发生变化的铁观音茶树被聚为同一亚类，且这两个亚类在同一类群中距离最远。3、基于ISSR分子标记的铁观音茶树分子指纹图谱构建基于11对扩增效果较好的ISSR引物的扩增产物多样性和特异性分析，从中筛选出5对核心引物，对其扩增产物的电泳图谱进行编码并组合，构建了13份供试茶树种质的分子指纹图谱，应用编码图谱可对供试茶树进行鉴别。基于扩增谱带的特异性，引物UBC-843可将供试种质中的9份铁观音茶树与其他近缘茶树进行区分，引物UBC-857可以将两份种性纯度较高的铁观音茶树与其他发生性状分化的茶树种质鉴别出来。4、不同表型性状铁观音茶树的基因表达差异分析以种质性状纯度较高的和发生明显表型性状分化的两份铁观音茶树为材料（分别命名为铁观音茶树X和Y），应用cDNA-AFLP技术，分析了两份茶树样本基因表达的差异性，结果表明：以256对AFLP引物组合对样品进行扩增，在两份样本之间共分离出75条扩增稳定的差异条带，其中36条在铁观音茶树X上高表达，39条在铁观音茶树Y上高表达。分离回收的差异条带进行克隆转化测序后获得63条有效序列，经BlastX比对后得出51条差异序列具有同源相似序列，其中17条与物质合成代谢功能相关，11条和转录调控功能相关，3条与信号转导相关，3条与物质输送与传递相关，17条功能未知。5、不同表型性状铁观音茶树差异表达基因的荧光定量表达应用实时荧光定量PCR技术，对不同表型性状的铁观音茶树样本之间的10条差异表达基因，进行了表达量的定量分析。结果表明：10条基因在两份茶树样本之间的表达量差异明显，其中有8条基因在铁观音茶树Y上表达量较高，相对表达量在1.11-5.56之间，其余两条基因在铁观音茶树Y相对表达量分别为0.74和0.48。差异表达的基因可能与两份铁观音茶树在芽叶颜色、树姿、叶形和叶片着生状态等表型性状上的差异有关。更多还原

【Abstract】 Tie Guanyin is a national improved tea cultivar which isoriginally planted in Anxi County, Fujian. It is also a good breedingoriginal material in tea breeding. In recent years, some germplasmcharacters differentiations were found in Tie Guanyin tea treesduring cultivation, especially about the phenotypic characters.Maintaining the variety purity and economic characters of Tie Guanyintea trees were affected by these differentiations. In this research，the morphological variation of Tie Guanyin tea tree was studied, andthe otherness of Tie Guanyin tea trees with different phenotypiccharacter were studied by ISSR technique, and the molecularfingerprint of Tie Guanyin tea tree was established as well. cDNA-AFLP was used to study the gene expression difference between twokinds of Tie Guanyin tea trees with typical phenotypic difference.Themain results were as follows：1. Phenotypic character differentiations among differentTie Guanyin tea trees.Some morphological features of80Tie Guanyin tea trees wereinvestigated. The variability of the posture, bud-leaf color, serratenumber of leaf margin, lateral vein number, leaf thickness, and leaflength and leaf width among these trees were analyzed. The resultsshowed that the posture of the tea tree had the biggest variabilitywhich variable coefficient was48.95%. The second was the bud-leafcolor which variable coefficient was36.83%. Three principalcomponents were picked up by using principal component analysis,which could be used as reference indexes in distinguishing thephenotypic character differentiation of Tie Guanyin tea trees.Genetic similarity analysis and cluster analysis also indicated that the tea trees with higher variety purity had higher geneticsimilarity, and could be clustered in one group.2、The molecular markers differences of different TieGuanyin tea treesThe ISSR amplification reaction system was established andoptimized. The genetic diversity and relationship of thirteen teatrees were investigated by using ISSR. Ninety-one bands weregenerated by PCR using eleven primers, of which45bands werepolymorphic bands, and the polymorphic rate was49.45%. The geneticdiversity index of Nei and Shannon information index of thesethirteen tea germplasms were0.17and0.25, respectively, whichindicated that the genetic diversity level was low among thesesamples. Genetic similarity analysis indicated that the geneticsimilarity coefficient of13tea germplasms ranged from0.687to0.986, with average was0.805. This suggested that the geneticrelationships among these cultivars were close. The thirteen teagermplasms were divided into three groups by cluster analysis, nineTie Guanyin tea trees were clustered in one group, in which the twotrees with higher variety purity were clustered in one sub-group, theother two with variation phenotypic characters were clustered inanother sub-group, but the cluster distance between these two sub-groups was the furthest in the group.3. Molecular fingerprint of Tie Guanyin tea treeFive primers were selected from eleven primers to establish themolecular fingerprint of these thirty tea germplasms, which could beused to identify the samples. The primer UBC-843could be used toidentify the nine Tie Guanyin tea trees from allthe13tea trees, and primer UBC-857could distinguish the two tea trees with highervariety purity from the other trees with characters differentiation.4. The difference of gene expression between two differentphenotyp-es of Tie Guanyin tea treescDNA-AFLP technique was used to identify the two differentphenotype Tie Guanyin tea trees, which one named as Tie Guanyin teatree X and the other named as Tie Guanyin tea tree Y. Total75differently expressed bands were separated by using256pairs of AFLPprimers, of which36bands were higher expressed in Tie Guanyin teatree X, and the other39bands were higher expressed in Tie Guanyintea tree Y. Sixty-three bands were separated from these75bands andbe clone. BlastX comparison results indicated51of these sequenceswere homologous sequences,17sequences of these were related tomaterial synthesis and metabolism,11sequences were related totranscription-regulation,3sequences were related to signaltransduction,3sequences were related to the material delivery, and17sequences were unclear functional.5. qPCR of the differential expressed genes between twoselected Tie Guanyin tea treesTen differential expressed genes were chose to analyze theirexpressed quantity between Tie Guanyin tea tree X and Y by using qPCR.The results showed that there were obvious differences between thesetwo samples. Eight of these ten genes were higher expressed in TieGuanyin tea tree Y, and the relative expressed quantity ranged from1.11to5.56. The expression quantities of the other two genes in TieGuanyin tea tree Y were0.74and0.48. These expression differenceswere probable involved to the difference of bud-leaf color, treeposture, leaf morphology and epiphyllous angle between the two teatrees.更多还原