台灣茶樹種原葉部性狀及DNA序列變異之探討

Studies on the Variations in Leaf Characters and DNA Sequences of Tea Germplasm in Taiwan

胡智益(Chih-Yi Hu)

指導教授 ： 林順福

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01291

摘要

本研究主要目的包括：（1）評估葉部性狀變異以供品種鑑別之參考，（2）建立茶樹品種之DNA指紋資料庫及品種鑑定流程，（3）以DNA分子層次分析茶樹種原之遺傳歧異性及親緣關係，及（4）探討DNA分子標誌鑑別成茶品種與混合品種之可行性。
根據132個種原葉部性狀的調查結果，7個質的性狀（芽色、成熟葉色、葉形、葉片尖端形態及基部形態、芽葉茸毛、葉緣鋸齒形態）除了葉形性狀分佈較均勻外，其餘6個性狀均偏向於特定外表型；所調查8個數量性狀（成熟葉長、成熟葉寬、成熟葉厚、節間長度、成熟葉面積、成熟葉長寬比、葉緣齒數及葉脈角度）中，葉厚性狀及成熟葉長寬比的變異程度較小。以15個質與量的葉性狀部利用UPGMA法繪製的群聚分析圖，主要依據葉形及葉尖形態將茶樹種原分成三大群。
由100個ISSR引子篩選得到12條引子，共可產生67個訊號較強且具有明顯再現性的多型性條帶，此外，利用這些分子標誌建立133個茶樹種原之DNA指紋資料，並評估其遺傳歧異性，依據UPGMA進行群聚分析，可將所有茶樹種原區分成六大群，包括油茶群、赤芽山茶群、野生茶樹群、大葉變種與小葉變種混合群、大葉、小葉及大葉、小葉雜交種混合群及小葉變種群，並發現香櫞與香耳、大南灣白毛猴與黑毛猴、青心烏龍與烏金、早種與黑面早種、白葉與大冇、枝蘭與文山枝蘭及七堵白種與楓子林等具有極高之分子標誌相似性。在主成分向量分析方面，顯現的結果與利用群聚分析得到的親緣關係樹狀圖結果相符合。此外，ISSR分析說明台灣茶樹種原間仍存在相當大的遺傳歧異度（58%），但總遺傳歧異度雖大，高比例的遺傳歧異度是由台灣的野生茶樹所貢獻，重要品種間的相似性極高。針對台灣栽培面積較大的22個茶樹種原及3個新品種的ISSR DNA指紋資料可由3個引子（818、864及866）中的10個分子標誌得到有效鑑別。
在葉綠體序列分析方面，5組通用引子中，4組具有擴增產物，在總分析長度為4415 bp中，共得到23個序列變異區域，包含17個單一核苷酸變異（SNPs）及6個核苷酸插入或缺失（Indels），總變異長度為47 bp，可知品種間葉綠體DNA序列變異以SNP為主。在15個茶樹種原比較下，變異發生率為1.06％，顯示葉綠體DNA序列變異可利用於品種鑑別及母本來源追蹤。在粒線體序列分析方面， 8組通用引子中，6組引子具有擴增產物，在總分析長度為7164 bp時，共得到24個序列變異區域，其中包含9個SNPs及15個Indels，總變異長度為87 bp，可知品種間粒線體DNA序列變異以Indel為主。在15個茶樹種原比較下，序列變異發生率為1.21％，與葉綠體之變異程度相近。利用葉綠體及粒線體序列分析所產生的群聚關係結果類似，皆可大致區分成三大群，唯台灣山茶的分群結果略有差異。根據胞器序列分析的結果，緬甸是台茶18號的母本，但此次結果卻說明台茶18號的胞器序列比較類似Shan而非緬甸。結合葉部性狀、ISSR分析及胞器序列分析，說明香櫞、香耳及皋盧極有可能屬於小葉變種，其葉面積可能是因三者皆為多倍體植株所造成；此外，赤芽山茶類應與茶樹不同之物種。
在成茶品種鑑定方面，利用不同發酵程度的茶類探討製茶過程對DNA品質之影響，說明高溫（250℃）殺菁對成茶DNA的破壞力最強。利用成茶DNA樣品進行PCR擴增反應，說明位於分子量小於1000 bp的ISSR分子標誌之條帶強度變化不大且較穩定，應可利用成為辨識品種的依據；而利用胞器通用引子觀察所有包種茶的樣品均可得到穩定的專一性條帶，說明專一性條帶除了條帶強度較強、穩定性更高外，分子標誌可出現在較高分子量的位置，可應用於成茶的品種鑑定，又由於茶樹胞器基因體為單倍體，可有效利用於鑑定品種內變異或混合品種之茶葉。

關鍵字

茶樹 ； 葉部性狀 ； 葉綠體DNA序列 ； ISSR分子標誌 ； 粒線體DNA序列 ； 成茶品種鑑定

並列摘要

The main purposes of this study were: (1) to evaluate variation in leaf traits for variety identification, (2) to establish DNA fingerprints and a process for variety identification of tea, (3) to analyze the genetic diversity and/or parentage among germplasm of tea, and (4) to evaluate the feasibility of molecular identification for processed tea and tea mixtures from different varieties.
According to the leaf traits investigated from 132 tea germplasm, the distribution of 7 qualitative traits, including bud color, leaf color, leaf shape, apex shape, base shape, bud hair and morphology of leaf teeth, except for leaf shape, were subjected to specific phenotypes. The quantitative traits, including leaf length, leaf width, leaf thickness, internode length, leaf area, length/width ratio, number of leaf teeth and angle of vein, leaf thickness, and length/ width ratio of mature leaf exhibited relatively low variation. By using UPGMA method, the cluster diagram based on 15 quantitative and qualitative leaf traits showed that the tea varieties could be divided into three groups according to the leaf shape and apex shape.
Sixty-seven polymorphic bands with strong signal were amplified from 12 ISSR primers, which were prescreened from 100 primers. Furthermore, these 67 ISSR markers were used to develop the DNA fingerprints and analyze genetic diversity of tea germplasm. According to UPGMA method, the tea gerplasm could be divided into 6 groups: including C. tenuifolia group, Chyh Ya Sun Cha group, wild tea plants group, var. assamica and var. sinensis group, var. assamica with var. sinensis and their hybrid group and var. sinensis group. Some germplasm had high similarity in ISSR markers to other one, including Shiang Yuan and Shiang Eel, Dah Nan Wan Bair Mau Hour and Heh Mau Hour, Chin Shin Oolong and Wu Jin, Tzao joong and Chin Shin Tzao joong, Bair Yeh and Dah Pan, Jy Lan and Wen Sun Jy Lan, Chuh Du Bair Joong and Feng Tzy Lin.
Similar grouping result was found in the principal coordination components analysis and the cluster diagram. Furthermore, the ISSR analysis revealed that there is quite large genetic diversity among tea germplasm in Taiwan (58%). Despite the high genetic diversity among germplasm, high percentage of it was contributed by the wild tea plants, and most important cultivars had high degree of similarity. The most important 22 cultivars and 3 newly bred varieties could be efficiently identified with ten ISSR markers amplified by three primers (818, 864 and 866).
In the chloroplast DNA sequence analysis, 4 out of 5 primers could generate amplification products. There were 23 sites detected with DNA sequence variation in a total length of 4415 bp, including 17 single nucleotide polymorphism(SNPs) and 6 nucleotide insertion or deletion(Indels). The total variation length is 47 bp and the rate of variation is 1.06% in the survey of 15 tea germplasm. Therefore, the variation of chloroplast DNA sequences among germplasm is mainly SNPs , and it could be used to identify cultivars and to trace their female parents. In the mitochondria DNA sequence analysis, 6 of 8 primers could generate amplification products. There were 24 sites detected with DNA sequence variation in a total of 4415 bp length, including 9 SNPs and 15 Indels. The total variation length is 87 bp and the rate of variation is 1.21% in the comparison of 15 tea cultivars (similar to chloroplast). Therefore, the variation of mitochondrial DNA sequences among germplasm is mainly Indels. Similar result of cluster analysis was observed in chloroplast and mitochondria DNA sequences. Fifteen tea germplasm were classified into three groups, only Taiwan Sun Cha was done differently. According to the cytosomic sequences analysis, the sequence of TTES No. 18, resembled Shan instead of its female parent, Burma. Based on the leaf trait, ISSR DNA and cytosomic sequences analysis, Shiang Yuan, Shiang Eel and Gau Lu were identified as var. sinensis. And their larger leaf size was due to triploid. Besides, Chyh Ya Sun Cha might not be the same with Camellia sinensis.
Different levels of fermented tea were investigated on the isolated DNA quality. The result indicated that high temperature (250℃) was the most devastating step to the DNA in processed tea. The bands of amplified ISSR DNA under 1000 bp had stable signals, and they could be used in cultivar identification. On the other hand, stable and specific bands with higher molecular weight were obtained by using cytosomic primers for all. It suggestes that applying DNA techniques in cultivar identification of processed tea is feasible. Furthermore, the tea mixtures from different varieties could be identified by cytosomic DNA sequences because the cytoplasmic genome is haploid with unique sequence.

並列關鍵字

Tea ； mitochondria DNA sequence ； ISSR ； chloroplast DNA sequence ； variety identification of processed tea ； leaf character

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