本研究利用PCR-amplified sequencing及ISSR等技術，以獲取台灣杉(Taiwania cryptomerioides Hayata)的分子標誌(molecular marker)。並進一步探討台灣杉族群的遺傳多樣性 (genetic diversity) ，以及台灣杉與禿杉 (Taiwania flousiana Gaussen) 間的分子親緣關係 (molecular phylogeny) 。在PCR- amplified sequencing的分析方面，針對演化過程中變異快速的核糖體DNA (ribosomal DNA) 的內轉錄間隔區 (internal transcribed spacer, ITS) 進行序列分析，總計分析來自於台灣8個不同族群的108個台灣杉樣本，以及來自中國雲南的12個禿杉樣本。結果顯示台灣杉的個體內ITS序列具異質性 (heterogeneity) ，各族群的核&#33527;酸歧異度 (nucleotide diversity) 亦高，平均為0.18153 ( π&#20516;)。經中性檢測 (Tajima’s D test) ，發現台灣杉的ITS在演化的過程中，序列之變異符合中性假說。在禿杉樣本的分析也發現個體內ITS具歧異性、高的π&#20516; (0.19751) 以及符合中性假說。然後將ITS序列進行群叢分析 (clustering analysis) 顯示台灣杉各族群間並無明顯分群。台灣杉與禿杉間亦沒有明顯分群。經Fst&#20516;的分析，發現台灣杉各族群間之Fst&#20516;均小於0.05，而台灣杉與禿杉之間有部分程度的分化 (Fst&#20516;0.0441~ 0.0856，平均為0.0611) 。在ISSR分析方面，經篩選100條ISSR引子，其中有4個引子的效果較好，可以獲得具有再現性條帶。總計可獲得24個條帶，其中有17個條帶具有多型性 (polymorphic) 。將上述分子資料進行遺傳距離換算及群叢分析，發現台灣杉各族群間及台灣杉與禿杉間並無明顯的分群。以AMOVA進一步分析台灣杉8個族群間及與雲南禿杉族群樣本的差異性，發現台灣杉與禿杉種間的變異佔38.54％ (P < 0.001)，而種內的變異則佔61.46％ (P < 0.001)，此表示種內的變異大於種間的變異程度。而在台灣杉族群間分析的結果，族群間的變異佔總變異的15.26％ (P < 0.001)，族群內的變異佔84.74％ (P < 0.001)，分析的結果顯示群內的變方成分 (variance component) 大於群間的變方成分。綜合上述的分子資料發現，台灣杉族群間無明顯的分化且基因流傳頻繁，其遺傳變異存在於族群內，而族群間則無顯著的差異。在台灣杉與禿杉之間，則因分離已有一段時間，呈現有些微程度的分化，但尚未達兩不同種。

The aim of this study was to obtain the molecular marker of Taiwania cryptomerioides Hayata based on DNA sequence data of PCR- sequencing and inter-simple sequence repeat (ISSR), and to evaluate the genetic diversity of populations of Taiwania cryptomerioides Hayata and molecular phylogeny of T. cryptomerioides and Taiwania flousiana Gaussen. The sequence data based on the internal transcribed spacer (ITS) of a total of 108 samples of T. cryptomerioides were determined. Eight different populations of T. cryptomerioides and 12 samples of T. flousiana from Yunnan, China were analyzed. The finding of the study showed that heterogeneity of ITS region within individuals of T. cryptomerioides was high by showing high nucleotide diversity among ITS sequences both in T. cryptomerioides ( π = 0.18153) and T. flousiana ( π = 0.19751). The findings fit in Tajima’s D test of neutrality based on DNA sequence variation in the ITS region of T. cryptomerioides and T. flousiana. It is not obvious to incorporate into different population through clustering analysis based on data of the ITS region of T. cryptomerioides and T. flousiana. However, slightly genetic differentiation between T. cryptomerioides and T. flousiana was found, which figured of Fst (Fst = 0.0441~ 0.0856, an average value = 0.0611). On the other hand, the samples were studied by using ISSR markers. Of the 100 primers screened, 4 produced highly reproducible ISSR bands, and 24 discernible DNA fragments were generated with 17 being polymorphic. Based on cluster analysis of molecular data, the cluster is not clear among populations of T. cryptomerioides and T. flousiana. The analysis of AMOVA revealed that the variance component between species of T. cryptomerioides and T. flousiana was 38.54％ (P < 0.001); however, the variance component within species is 61.46 (P < 0.001). The variation within population of T. cryptomerioides was 84.74％ (P < 0.001) and the variance between populations is 15.26％ (P < 0.001), indicating that the genetic diversity of individuals within population was high. The aforementioned data suggest that gene flow among different populations of T. cryptomerioides was high, indicating that the genetic diversity was high among individuals of T. cryptomerioides but was low between populations. Furthermore, it is concluded both species are genetically closer and could be grouped into the same species.

第一章 前言.........................................................1
一、台灣杉 (Taiwania cryptomerioides Hayata)簡介....................1
二、核糖體DNA (rDNA) 相關研究.......................................4
三、ISSR技術原理及其應用............................................7
四、研究目的........................................................10
第二章 材料與方法...................................................11
一、採樣............................................................11
二、基因體DNA (genomic DNA) 的萃取..................................13
三、定量DNA.........................................................14
四、聚合&#37238;鏈鎖反應 (polymerase chain reaction，PCR).................15
五、DNA純化.........................................................20
六、DNA接合作用 （ligation）........................................21
七、轉形作用（transformation）......................................21
八、菌落篩選........................................................22
九、質體純化........................................................22
十、質體限制&#37238;切割及核酸定序........................................23
十一、ISSR條帶的電泳、染色及照相....................................24
十二、ITS序列分析...................................................25
十三、ISSR條帶分析..................................................28
第三章 結果.........................................................31
一、ITS序列的分析...................................................31
二、族群內與族群間遺傳變異的分析....................................32
三、ISSR分析........................................................38
四、歸群分析的結果..................................................44
第四章 討論.........................................................49
第五章 結論.........................................................57
參考文獻............................................................59
附錄一..............................................................66

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