茶樹品種分子鑑定技術之開發及遺傳圖譜之建構

Development of DNA Markers for Variety Identification and Construction of Genetic Maps in Tea

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/博士(2013年)

https://doi.org/10.6342/NTU.2013.03059

摘要

本研究主要目的包括：（1）開發臺灣茶樹經濟栽培品種及其商品（成品茶）的分子鑑定技術，作為保障商業品種之智慧財產權的客觀依據，（2）應用DNA分子標誌分析臺灣茶樹種原之遺傳歧異度及親緣關係，作為茶樹雜交育種之親本選擇之參考，及（3）建構整合雌親與雄親之茶樹遺傳圖譜，以供未來分子育種之用。
本研究開發穩定、快速而可靠的EST-SSR、STS及CAPS分子標誌，以鑑定臺灣12個茶樹主要經濟栽培品種。開發41組EST-SSR引子，共偵測253個對偶基因，平均為6.17個對偶基因；而其多型性資訊量（PIC）介於0.19∼0.86間，平均為0.64，表示多型性相當豐富。僅需本研究所開發之兩組EST-SSR引子，即可完全鑑定臺灣12個主要經濟栽培品種。由茶樹細胞質基因體及EST序列設計2組STS及37組CAPS分子標誌，其中選用粒線體M02、葉綠體C02、核內G01、G03及G04等5個核心分子標誌，足以有效鑑別12個臺灣茶樹主要經濟栽培品種。此外，本研究所開發EST-SSR、STS及CAPS分子標誌，尚可適用於臺灣主要生產的特色茶（包括部分發酵茶類及綠茶類）之成品茶的分子鑑定，另由於具有共顯性特性，亦可作為追蹤品種之父母本來源的工具。
根據2組STS及37組CAPS分子標誌在55個臺灣茶樹重要種原之遺傳歧異度分析、主成分向量分析及群聚分析結果，可區分成小葉變種群（Camellia sinensis var. sinensis）、大葉變種群（C. sinensis var. assamica）及野生茶樹群（C. formosensis），小葉變種群及大葉變種群的遺傳歧異度明顯高於野生茶樹群。但在臺灣的經濟栽培品種中，栽培面積最大品種之青心烏龍與第三大品種四季春的血緣接近，且新育成的臺茶系列品種多具有青心烏龍的血統，因此，擴大臺灣茶樹栽培品種的遺傳歧異度，將是未來臺灣茶樹育種的重要方向。
茶樹受限於自交不親和性及幼苗至成熟植株需要多年時間培育和評估，以選拔優良單株，故育成茶樹新品種通常需要超過20年以上。遺傳圖譜是遺傳育種研究的重要工具，本研究利用兩個經濟栽培品種（臺茶19號及臺茶8號）的F1雜交後裔建構整合雌親與雄親之茶樹遺傳圖譜。在篩選得到574個SSR、CAPS、AFLP、ISSR及RAPD等多型性分子標誌中， 69個為高度偏差分離之多型性分子標誌（P < 0.001）（12.0%）未加入後續分析。而在參與建構圖譜的505個分子標誌，包含265個來自雄親（52.5%）、163個來自雌親（32.3%）、65個來自雙親的異接合顯性標誌（12.9%），及12個共顯性標誌（2.4%），其中共顯性標誌及來自雙親的異接合顯性標誌扮演連接雄親與雌親圖譜的橋樑基因座。最終的遺傳圖譜是由367個連鎖標誌包含36個SSR標誌、3個CAPS標誌、1個STS標誌、250個AFLP標誌、13個ISSR標誌及64個RAPD標誌所建構出18個連鎖群，圖譜總長為4482.9 cM，圖譜密度為12.2 cM。本研究建構的圖譜是目前全世界基因體覆蓋率最高之茶樹的遺傳圖譜，可應用於比較定位研究、數量性狀基因座定位及分子標誌輔助選種。

關鍵字

茶樹 ； 成品茶 ； 品種鑑定 ； 遺傳歧異度 ； 遺傳圖譜 ； DNA分子標誌 ； EST-SSR分子標誌 ； STS分子標誌 ； CAPS分子標誌

並列摘要

The main purposes of this study are: (1) to develop DNA markers for identifying tea varieties and commercial product (processed tea) in Taiwan that can be utilized for protecting the intellectual property rights of the commercial varieties, (2) to analyze the genetic diversity and phylogenetic relationship among tea germplasm in Taiwan by DNA markers, which can be used as a reference for parental selection of hybridization breeding, and (3) to construct an integrated genetic map of tea that can be applied for molecular breeding in the future.
In this study, EST-SSR, STS and CAPS markers are developed as the stable, fast and reliable tools for identifying 12 prevailing tea varieties in Taiwan. Of the developed 41 EST-SSR primer pairs, there are 253 alleles identified with an average of 6.17 alleles per primer pair, and the polymorphism information content (PIC) ranges from 0.19 to 0.86, with an average of 0.64. Based upon the PIC values, it indicates that these markers have high polymorphism. Besides, it is sufficient to distinguish 12 prevailing tea cultivars in Taiwan by using two EST-SSR primer pairs. Moreover, two STS and 37 CAPS markers derived from cytoplasmic genome and ESTs of tea have been developed in this study. For effectively identifying 12 prevailing tea cultivars in Taiwan, five core markers were developed, including M02 (mitochondria), C02 (chloroplast), G01, G03 and G04 (nuclear). All of these markers in this study are used not only for identification of prevailing tea cultivars in Taiwan but also for processed tea, including green, Paochong and Oolong tea. Otherwise, these markers could be used to trace the parents of cultivars due to the co-dominant inheritance.
Based on the genetic diversity, principal coordinates, and cluster analyses of two STS and 37 CAPS markers, 55 tea germplasm collected in Taiwan consist of three main groups: sinensis type (Camellia sinensis var. sinensis), assamica type (C. sinensis var. assamica) and wild species in Taiwan (C. formosensis). The sinensis type and assamica type performed greater genetic diversity than the wild species. However, the genetic distance between the first (Chin-Shin-Oolong) and the third (Shy-Jih-Chuen) prevailing cultivars is very close, and many recently developed varieties are the descents of Chin-Shin-Oolong. It implies the necessity of incorporating wild species in the breeding programs to expand the genetic diversity of tea cultivars in Taiwan.
The genetic study on important traits of tea is difficult because of the self-incompatibility in nature. Moreover, the development of a new variety usually takes more than 20 years, including many years from seedling to mature plants for trait investigation. A genetic map is an essential tool for genetic study and breeding. In this study, we have developed an integrated genetic map of tea (Camellia sinensis) by using a segregating F1 population derived from a cross between two commercial cultivars (‘TTES 19’ and ‘TTES 8’). A total of 574 polymorphic markers (including SSR, CAPS, STS, AFLP, ISSR and RAPD) were identified, and 69 markers with highly significant levels of segregation distortion (P < 0.001) (12.0%) were excluded from further analyses. Of the 505 mapped markers, there were 265 paternal markers (52.5%), 163 maternal markers (32.3%), 65 doubly heterozygous dominant markers (12.9%), and 12 co-dominant markers (2.4%). The co-dominant markers and doubly heterozygous dominant markers were used as bridge loci for the integration of the paternal and maternal maps. The integrated map comprised 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR and 64 RAPD that were assigned to 18 linkage groups. The linkage groups represent a total map length of 4482.9 cM with the density of 12.2 cM. This genetic map has had the highest genetic coverage so far, which could be applied to comparative mapping, QTL mapping and marker assisted selection (MAS) in the future.

並列關鍵字

Tea (Camellia sinensis) ； Processed tea ； Variety identification ； Genetic diversity ； Genetic map (linkage map) ； DNA marker ； EST-SSR marker ； STS marker ； CAPS marker

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