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Molecular characterization of the rice mutant SA0420 derived from Tainung 67 associating leaf aroma applying Amplified Fragment Length Polymorphism
水稻(Oryza sativa)依據香味的有無，可以分為香米（aromatic rice）以及非香米（non-aromatic rice），由於具有特殊的香氣以及口感，香米很受全世界米食的消費者喜愛。AFLP（amplified fragment length polymorphism）的分子標誌已被廣泛應用在基因研究，主要是因為AFLP技術具有分析multiple-allele的能力、高再現性（reproducibility）以及專一性。本論文是利用AFLP分析非香米品種水稻台農67號（TNG67）以及香米突變體SA0420的香味基因，配合AFLP/BSA分析F2子代（SA0420 x TNG67）以及重組自交系族群（recombinant inbreed lines; RILs），最後選殖到6個與香味性狀有關的多型性片段（polymorphic fragments）。由於水稻具有豐富的基因組資料庫，因此可以利用該資料庫上基因組的資訊與多型性片段序列比對的結果，預測水稻香味的候選區域，並且根據連鎖失衡（linkage disequilibrium）的原理，利用SSR標誌篩選所有的候選區域，最後在水稻第6條染色體的BAC找到極有可能參與水稻香味的調控的區域。另外，將選殖的6個AFLP多型性片段設計成簡易的PCR-based標誌STS（sequence tagged sites），進行SCAR(sequence characterized amplified region)分析F2，並配合SSR標誌分析F2的結果，利用Qgene和Mapmaker分析並建立香味基因的分子標誌圖譜，最後標誌E-AG/M -CTT-178-STS與水稻香味有極顯著的相關，而且該標誌與第2條染色體的SSR標誌RM262、RM106為同一個連鎖群，顯示在該位置也與香味的調控有關。未來，可以針對水稻第2條和第6條染色體的兩個區域，利用據圖選殖的原理將可以有效的選殖水稻的香味基因，並將香味基因轉移到其他優良品系以提升水稻的米質。並且利用AFLP標誌E-AG/M -CTT-178 -STS作為未來水稻育種時，分子標誌的輔助選拔（marker-assisted selection, MAS）的依據，解決早期篩選的問題，加快國內水稻香味育種的效率。
Rice(Oryza sativa)can be classified as aromatic rice and non-aromatic rice according to the fragrance. Aromatic rice are preferred by the consumers all over the world, due to its unique flavour and palatability. AFLP (amplified fragment length polymorphism) markers were broadly used to establish the genetic linkage map of the target gene. Because AFLP technique can perform multiple-allele analysis simultaneously, great reproducibility and specificity, these advantages with regard to genome mapping make it better than than RAPD. Rice genome had been completed and constructing high-density marker map will facilitate to clone candidate genes by map-based cloning approach. In this study, the rice cultivar TNG67 (non-aromatic rice), SA0420 (aroma mutant line), F2 population (SA0420 x TNG67), and RILs (recombinant inbreed lines) were analyzed by AFLP/BSA technique. A total of six polymorphic fragments which related to rice aroma were cloned, and sequenced. Because of the highly rich information of rice genome database available, it is very efficient to predict the candidate regions controlled rice aroma by sequencing alignment. In addition, according the linkage disequilibrium, candidate regions could be confirmed by closely linked marker, such as rice SSR markers. Finally, on rice chromosome 6, two BAC clones were detected to associate with a rice aroma gene. Besides, six polymorphic fragments were designed as PCR-based markers; STS, to analysis the F2 population by SCAR (sequence characterized amplified region) and some SSR markers were also used to detect the linage and established a linkage map. Consequently, marker E-AG/M -CTT-178 -STS was found to be related to rice aroma, and this marker was assigned to the same linkage group as chromosome 2 between SSR marker RM262 and RM106. In the future, the two regions, chromosome 2 and chromosome 6, will be focused to isolate the rice aroma candidate genes by map-based cloning approach, and to transfer aroma genes into elite rice cultivars to improve rice quality. Those markers linked with rice aroma genes will be useful for MAS (marker-assisted selection) in rice breeding.
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