Studies of genetic diversity and the establish of the molecular data base of Taiwan tea cultivars 顏呈恩、李世傑
E-mail: [email protected]
ABSTRACT
In this study, different tea varieties preserved in Taitung Tea Research and Extension Station (TTES) were used as materials. Tea leaves were manufactured to green tea and 10 kinds of tea catechins were determined by HPLC, and to study the genes Diversity based on these chemical markers. Principal component analysis and UPGMA clustering analysis based on the contents of tea catechins have been used in the study of correlations between manufacturing suitabilities and agronomic traits. Molecular markers, including the nucleotide sequences of pITS2, trnL intron, trnL-trnF IGS were also used in determining the genetic diversity of teas preserved in Tea Research and Extension Station of tea in Taiwan. Our results have shown that the breed tea varieties are generally have higher total catechin content. TTES No.4 has the highest methylation catechins of 3.3 tea (g/100g d.w.), with the greatest potential used in anti-allergy products, for all the tea species tested. Catechins contents in wild Camellia speciesis generally low. Even though these indigenous tea species are much more resist to the environment stress. Unfortunately, more or less negative agronomic traits will be carriered. Total catechins (TC) content is correlated to the contents of caffeine, EC, ECG, EGC, EGCG and four methylated catechin content. Caffeine’s content is correlated to the content of EGCG, ECG. Principal component analysis (PCA) results have shown that Taiwan wild camellia Tea fell the third and fourth quadrant. Taiwan wild camellia Tea is best for making black tea, and most of the tea cultivars located in third and fourth quadrant are suitable for manufacturing black tea too. For those tea cultivars suitable for manufacturing green tea are scattering in all four quadrantswith overlapping some cultivars suitable for manufacturing Fermented tea. PCA results also indicated that the content and distribution of catechins are correlated with the tea varieties and the manufacturing suitablility and are valuable data for future tea breeding reference. The higher content of caffeine and EGCG are related to the capacities ofinsect resistance of tea, such as anti-mite, anti-leaf beetle, anti-thrips and leaf beetle resistance. Cluster analysis for tea varieties based on UPGMA method using MVSP software has separated two wild Camellia Tea (TD85 and TD100) can be species with other into an independent cluster, showing their genetic pecificity. Single nucleotide polymorphisms were observed based on three multiple sequence alignments of pITS2 sequence of 112 tea varieties, 104 trnL intron sequences, and 98 trnL-trnF IGS sequencespITS2 has the highest nucleotide sequence polymorphism with sequence similarity between 0.379 ~ 0.994, and up to 149 variable sites. Two cpDNA sequences are relatively conserved in these two area, with sequence similarity of 0.948~1.000 for trnL intron and sequence similarity of 0.979 to 1.000 for trnL-trnF IGS. The sequence variation of pITS2 can be used in establishing the molecular fingerprinting database for Taiwan tea plants. Phylogenetic trees constructed based on nucleotide sequence of pITS2 using Neighbor Joining Method, Minimum Evolution methods and Mmaximum parsimony methods can clustered wild teas in Taiwan into two independent clustersalong with other five groups. Our results in genetic diversity is important in future tea breeding selection program.
Keywords : tea、catechins、molecular markers、germplasm resource、genetic diversity Table of Contents
封面內頁 簽名頁 中文摘要iii 英文摘要vi 誌謝viii 目錄ix 圖目錄xii 表目錄xiv 1.前言1 2.文獻回顧3 2.1茶葉簡史3 2.1.1茶葉簡 介3 2.1.2茶樹在生物學上的分類3 2.1.3臺灣茶樹簡史 5 2.2茶葉主要化學成分介紹8 2.2.1兒茶素8 2.2.2咖啡因9 2.2.3其他化學 成分13 2.2.4以化學成分分析遺傳變異性13 2.3臺灣茶樹樹種之分子鑑定技術14 2.3.1臺灣茶樹種原介紹17 2.3.2分子標記之應 用17 2.3.3細胞核內特定基因片段及葉綠體基因體的組成及其分子標誌之應用22 2.3.4DNA分子標記在臺灣茶樹品種資源研 究24 2.4建立臺灣茶樹樹種DNA指紋資料庫29 3.材料與方法31 3.1材料與儀器31 3.1.1實驗材料31 3.1.2兒茶素含量分析之實 驗藥品31 3.1.3兒茶素含量分析之儀器設備32 3.1.4萃取genomic DNA之實驗藥品33 3.1.5偵測DNA濃度之儀器設備34 3.2研究 方法39 3.2.1兒茶素測定39 3.2.2茶樹種原兒茶素及葉部性狀之分析39 3.2.3萃取Genomic DNA40 3.2.4引子設計41 3.2.5聚合?
鏈鎖反應43 3.2.5電泳分析46 3.2.6定序46 3.2.7臺灣茶種DNA資料庫之建立與序列分析46 4.結果與討論47 4.1以生化標誌將 臺灣茶種建立種原資料庫 47 4.1.1茶葉葉片中兒茶素的HPLC分析與定量47 4.1.2臺灣茶樹種原兒茶素差異及相關性分析60 4.1.3臺灣茶樹種原兒茶素與農藝性狀之相關性分析62 4.1.4茶樹品種間兒茶素的主成份分析65 4.1.5依據生化標誌將臺灣茶 樹種原進行群集分析71 4.2 以分子標誌將臺灣茶種建立種原資料庫 75 4.2.1pITS2序列分析75 4.2.2依據pITS2序列進行茶樹 種原之群集分析85 4.2.3cpDNA序列分析88 4.2.4依據cpDNA序列進行茶樹種原之群集分析102 5.結論108 參考文獻110 圖目 錄 圖2.1兒茶素結構圖10 圖2.2甲基化兒茶素結構圖11 圖2.3兒茶素前驅物與咖啡因結構圖12 圖3.1pITS2序列結構示意圖42
圖3.2trnL-trnF IGS與trnL intron序列結構示意圖43 圖4.1(-)-Gallocatechin（GC）和caffeine之檢量線48
圖4.2(-)-Epigallocatechin gallate（EGCG）和(-)-Epigallocatechin（EGC）之檢量線49 圖4.3(-)-Catechin（C）和(-)-Epicatechin
（EC）之檢量線50 圖4.4(-)-Epicatechin gallate（ECG）之檢量線51 圖4.5兒茶素、咖啡因和甲基化兒茶素HPLC圖譜52 圖4.6臺東茶葉改良場107種茶樹樹種編號對照之主成分散佈圖68 圖4.7臺東茶葉改良場107種茶樹種原類型主成分散佈圖69 圖4.8臺東茶葉改良場107種茶樹種親緣性原類型主成分散佈圖70 圖4.9以化學成分進行將臺灣茶樹樹種107種作群集分析所 得UPGMA圖73 圖4.10裁切pITS2序列示意圖，以臺茶12號為例76 圖4.11臺灣茶樹種原pITS2多重序列比對圖77 圖4.12臺灣 茶樹種原pITS2分子標誌群集分析圖86 圖4.13臺灣茶樹種原trnL intron多重序列比對圖90 圖4.14臺灣茶樹種原trnL-trnF IGS 多重序列比對圖97 圖4.15臺灣茶樹種原trnL intron分子標誌群集分析圖104 圖4.16臺灣茶樹種原trnL-trnF IGS分子標誌群集 分析圖106 表目錄 表2.1茶葉的分類及製法4 表2.2臺灣茶業改良場培育的茶種及其形態特徵表7 表3.1臺灣育成茶樹品種資料 表35 表3.2臺東茶業良場保存茶樹之品種資料表36 表3.3pITS2、trnL-trnF IGS與trnL intron PCR反應溶液配方44 表3.4pITS2 PCR溫度循環參數45 表3.5trnL-trnF IGS與trnL intron PCR溫度循環參數45 表4.1臺東茶業改良場107種茶樹品種化學成分分 析表53 表4.2不同茶樹品種兒茶素與咖啡因含量範圍59 表4.3臺東茶葉改良場107種茶葉生化成分之相關性61 表4.4兒茶素與 農藝性狀之相關性64 表4.5臺灣主要茶種10項化學成分之主成分分析特徵向量67 表4.6利用生化標誌得到107個茶樹種原群 集聚分群表74 表4.7臺灣茶樹種原pITS2序列相似度79 表4.8臺灣茶樹種原pITS2序列變異位點與核?酸比例表80 表4.9臺灣茶 樹種原trnL intron序列相似度89 表4.10臺灣茶樹種原trnL intron序列變異位點與核?酸比例表95 表4.11臺灣茶樹種原trnL-trnF IGS序列相似度96 表4.12臺灣茶樹種原trnL-trnF IGS序列變異位點與核?酸比例表101
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