- 學位論文
芭菲爾鞋蘭屬分子標誌的研究
Studies of the Molecular Markers for genus Paphiopedilum
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摘要
以聚合酶連鎖反應為核心的DNA分子標誌技術主要分成兩大類，一為偵測整個基因組變異，另一為針對目標DNA序列分析。在本研究中，以簡單序列重複分子指紋技術 (inter-simple sequence repeat, ISSR) 研究芭菲爾鞋蘭物種的分子指紋，同時也針對rDNA ITS以及葉綠體的基因間隔 (intergenic spacer, IGS) 進行分析；依據不同分子標誌技術的結果分別建立兩個區分芭菲爾鞋蘭屬的流程。 應用簡單序列重複分子指紋技術偵測芭菲爾鞋蘭物種整個基因組的變異，從加拿大哥倫比亞大學設計的第九組引子組中挑選了七個引子進行擴增反應，記錄30個芭菲爾鞋蘭物種的分子指紋，再以UPGMA (unweighted pair-group method using arithmetic average) 進行群叢分析，可將芭菲爾鞋蘭屬的物種分成六群，分別為Parvisepalum、Sigmatopetalum、Polyantha、Brachypetalum、Paphiopedilum以及Cochlopetalum，與Karasawa及Saito (1982) 的分類結果一致。 另一部份針對芭菲爾鞋蘭葉綠體的基因間隔trnS-trnM、trnR-trnN IGS以及rDNA ITS進行序列分析，並建構親緣關係樹狀圖。進行切割擴增多型性序列分析 (cleaved amplified polymorphic sequences, CAPS) ，以限制酶TspRI對trnS-trnM IGS進行酶切反應，可以區分芭菲爾鞋蘭的三個亞屬Parvisepalum、Brachypetalum及Paphiopedilum；以限制酶SfaNI切割rDNA ITS可以區分section Pardalopetalum以及Barbata。在trnR-trnN IGS的序列中發現在section Paphiopedilum以及Barbata具有一段140bp長度的缺失，最後根據trnR-trnN IGS的序列設計專一性引子進行擴增，可以將section Paphiopedilum以及Barbata區分出來。綜合以上的實驗結果，發展可以鑑別芭菲爾鞋蘭屬的流程。 從簡單序列重複分子指紋技術以及切割擴增多型性序列分析的結果可以將芭菲爾鞋蘭的物種分群，縮小研究的範圍。雖然芭菲爾鞋蘭屬的rDNA ITS以及葉綠體的基因間隔的差異性較小，在序列長度變化也不大，但是變性梯度膠體電泳可以區別相同長度DNA的些微差異。對ITS以及trnS-trnM IGS變異較大的片段設計引子進行擴增，共設計五個片段：ITS-a、ITS-b、SM1、SM2以及SM3，分別找出每個片段最適當的變性劑濃度，再對相同分群的物種進行變性梯度膠體電泳，從變性梯度膠體電泳的結果發現ITS-a具有較好的解析力，加上SM1的變性梯度膠體電泳圖，可以鑑定收集到的全部物種。
並列摘要
DNA molecular markers based on PCR are divided into two groups: one is to detect the whole genome variation and the other is to focus on target DNA sequence. In this research, Inter-simple sequence repeat (ISSR) was employed to study the molecular fingerprinting of Paphiopedilum genome and rDNA internal transcribed spacer (ITS) and chloroplast intergenic spacers (IGS) were selected to do phylogenetic analysis. According to the results of these different techniques, two processes were established for differentiation. ISSR technique was applied to detect the whole genome variation of 30 species in Paphiopedilum. Seven out of hundred primers from University of British Columbia (UBC) were selected. The unweighted pair-group method using arithmetic average (UPGMA) was employed to analyze the phylogenetic relationships based on ISSR data. The phylogenetic trees subdivide Paphiopedilum species into six groups. The result was consistent with the subgenus classification of Karasawa and Saito (1982), which include Parvisepalum, Brachypetalum, Paphiopedilum, Sigmatopetalum, Polyantha and Cochlopetalum. Another part of this study was to analyze the chloroplast intergenic spacers and rDNA ITS of genus Paphiopedilum such that the phylogenetic tree can be constructed. Cleaved Amplified Polymorphic Sequence (CAPS) was employed to study the polymorphism pattern of genus Paphiopedilum. The trnS-trnM IGS treated by TspRI could separate three subgenra: Parvisepalum, Brachypetalum and Paphiopedilum. The restriction pattern of rDNA ITS cleaved by SfaNI could separate sectionPardalopetalumand Barbata. There was a 140bp deletion in the sequence of trnR-trnN IGS of the section Paphiopedilum and Barbata. To further distinguish section Paphiopedilum and Barbata, specific primers were used to amplify trnR-trnN IGS. The results built a different system to differentiate genus Paphiopedilum. The variation regions of ITS and trnS-trnM IGS are selected for PCR amplification and the products were analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE can detect the small variation, even one nucleotide difference, in DNA sequence of the same length. Among five fragments: ITS-a, ITS-b, SM1, SM2 and SM3, ITS-a was found to exhibit a better resolution than others.
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