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研究生:沈姿秀
研究生(外文):Tzu-Hsiu Shen
論文名稱:台灣黃芩、南台灣黃芩之遺傳多樣性研究
論文名稱(外文):Genetic diversity of Scutellaria taiwanensis and S. austrotaiwanensis
指導教授:周昌弘周昌弘引用關係江友中江友中引用關係
指導教授(外文):Chang-Hung ChouYu-Chung Chiang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:99
中文關鍵詞:台灣黃芩南台灣黃芩遺傳歧異度單系群複系群譜系分類基因流傳隔離分化
外文關鍵詞:Scutellaria taiwanensisS. austrotaiwanensisgenetic diversityparaphylylineage-sortinggene flowisolation-by-distance
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唇形科黃芩屬的台灣黃芩(Scutellaria taiwanensis Wu)及南台灣黃芩(S. austrotaiwanensis Hsieh & Huang)為台灣特有種,前者現今僅分布於里龍山及浸水營,後者僅分布於里龍山,兩特有種因其生育環境受到嚴重破壞極需保護,同時亦為研究保育近緣物種遺傳多樣性的絕佳材料。本研究針對台灣黃芩及南台灣黃芩的野外族群,完成調查及取樣外,進而以分子標記估算其種內及族群間遺傳變異程度及分布,以了解此近緣稀有植物的遺傳歧異度及族群遺傳模式。利用葉綠體DNA之atpB-rbcL片段、粒線體DNA nad1 gene之intron 2區段及細胞核中核醣體DNA之ITS區間重建親緣關係,依葉綠體DNA與細胞核核醣體DNA結果顯示,兩物種各自形成單系群,而粒線體DNA資料則呈現複系群,造成三基因組呈現不一致的結果,推論可能是因為三基因組之變異速率不同,導致在種或族群的階層中各自停留在不同的譜系階段所呈現之結果。依據種間及族群間的基因流傳與族群分化程度結果,葉綠體DNA(Nm = 0.09)及細胞核核糖體DNA(Nm = 0.02)資料得知此二特有種間具有極低的基因流傳度,顯示此二近緣種間的基因流傳受到阻礙導致分離,推估粒線體DNA在族群間的相似性,並非來自族群間基因交流,而是隨機保留下來的祖先多型性。依Mantel test與歧點頻率分析的結果,顯示此二黃芩族群結構不符合隔離分化模式,其變動歷史亦不支持族群穩定成長狀態。由小族群以及低遺傳歧異度顯示此二特有物種正面臨嚴重的危機,根據分子資料結果,因其族群間之遺傳變異遠小於族群內之遺傳分化,顯示每個族群應該視為不同的管理單位。
Scutellaria taiwanensis and S. austrotaiwanensis are endemic and relative plants distributed in southern Taiwan, and are restricted in small regions located on the Lilungshan and Chingshuiying. Both species are good candidates to study on genetic structures and conservation genetics of endemic species complex. In this study, the gene genealogy of the cpDNA atpB-rbcL noncoding spacer, mtDNA nad1 b/c exons and nrDNA ITS region were reconstructed to assess the phylogeographic pattern of two closely related species, S. taiwanensis and S. austrotaiwanensis. Two network trees of the cpDNA and nrDNA data contributed by the minimum spanning network and neighbor-joining tree were identified two major clades, S. taiwanensis and S. austrotaiwanensis. The reconstructed neighbor-joining tree of the mtDNA data showed the paraphyly of each species, indicating the different lineage-sorting stage. In addition, there were low gene flow between S. austrotaiwanensis and S. taiwanensis in cpDNA (Nm = 0.09) and nrDNA (Nm= 0.02), indicating segregation between two closely related species and high levels of shared ancestral polymorphisms in mtDNA. The results of the Mental test and mismatch distribution analysis revealed that the distribution of genetic variation didn’t follow the "isolation by distance" model, and it wasn’t in a state of population growth either. Small population size and low level of genetic diversity reveal that the two rare species are under serious threats. Each population should be recognized as a different management unit.
目 錄

摘要.............................................I
Abstract.......................................III
謝誌.............................................V
目錄............................................VI
圖目錄...........................................X
表目錄.........................................XII
第壹章 緒言.....................................1
一、特有物種的保育與遺傳多樣性......................1
二、親緣地理學的發展及研究.........................4
三、分子標誌的發展與應用...........................5
四、分子技術在探討族群結構的應用....................7
五、台灣黃芩與南台灣黃芩的研究歷史回顧...............9
(一)台灣黃芩、南台灣黃芩的分類地位及形態特徵........9
(二)台灣黃芩、南台灣黃芩的分佈與現況..............11
六、研究動機與目的................................12
第貳章 材料與方法................................13
一、研究材料.....................................13
(一)野外採集...................................13
(二)總基因體DNA萃取和定量.......................14
二、實驗方法.....................................14
(一)引子設計...................................14
(二)聚合酶連鎖反應..............................15
(三)DNA純化....................................15
(四)基因選殖....................................15
1. 接合作用...................................15
2. 轉型作用...................................16
(五)萃取質體DNA.................................17
(六)DNA定序.....................................17
(七)資料分析....................................17
1. DNA序列整理與校對...........................17
2. 親緣分析...................................18
3. 遺傳歧異度分析..............................18
4. 中性假說測試................................19
5. 族群間核苷酸歧異度(dXY, dA)分析.............20
6. 族群遺傳分化指數(FST)及基因流傳值(Nm)分析......21
7. 歧點頻率分布(Mismatch Distribution)分析........22
8. Mantel test...................................23
第參章 結果......................................24
一、核苷酸多型性分析...............................24
(一)序列分析....................................24
(二)遺傳歧異度分析...............................26
(三)中性假說測試(neutrality test)..............27
二、族群遺傳多型性分析.............................28
(一)族群遺傳歧異度分析...........................28
(二)族群之中性假說測試...........................30
(三)族群間核苷酸歧異度(dXY , dA)分析............31
(四)族群遺傳分化指數(FST)及基因流傳值(Nm)分析...32
(五)歧點頻率分布(Mismatch Distribution)分析.....33
(六)Mantel test................................34
三、親緣關係圖的建構...............................34
(一)Neighbor-joining親緣關係樹...................34
(二)Minimum spanning network最小關聯網狀樹狀圖....35
第肆章 討論......................................37
一、胞器DNA序列資料................................37
(一)遺傳變異.....................................37
(二)遺傳歧異度...................................38
(三)中性假說測試.................................40
(四)遺傳分化及族群結構............................41
二、細胞核DNA序列資料..............................42
(一)遺傳變異與遺傳歧異度..........................42
(二)中性假說測試.................................43
(三)遺傳分化及族群結構............................44
三、胞器DNA與核DNA序列片段分析比較...................44
四、台灣黃芩與南台灣黃芩之保育.......................46
第伍章 結論.......................................48
參考文獻..........................................49
作者簡介..........................................99


圖 目 錄

圖1. 台灣黃芩與南台灣黃芩之形態特徵..................10
圖2. 台灣黃芩之植株照片.............................73
圖3. 南台灣黃芩之植株照片...........................74
圖4. 台灣黃芩與南台灣黃芩之分佈現況..................11
圖5. 台灣黃芩、南台灣黃芩與外群採集點................75
圖6. 根據葉綠體DNA資料以聚類分析法構築台灣黃芩與南台灣黃芩親緣樹狀圖........................76
圖7. 根據粒線體DNA資料以聚類分析法構築台灣黃芩與南台灣黃芩親緣樹狀圖........................77
圖8. 根據細胞核核糖體DNA資料以聚類分析法構築台灣黃芩與南台灣黃芩親緣樹狀圖...................78
圖9. 根據葉綠體DNA資料構築台灣黃芩與南台灣黃芩之MINSPNET網狀親緣關係圖......................79
圖10. 根據粒線體DNA資料構築台灣黃芩與南台灣黃芩之MINSPNET網狀親緣關係圖....................80
圖11. 根據細胞核核糖體DNA資料構築台灣黃芩與南台灣黃芩之MINSPNET網狀親緣關係圖...............81
圖12. 台灣黃芩與南台灣黃芩cpDNA歧點頻率分佈圖............82
圖13. 台灣黃芩與南台灣黃芩mtDNA歧點頻率分佈圖............83
圖14. 台灣黃芩與南台灣黃芩nrDNA歧點頻率分佈圖............84
圖15. 利用葉綠體DNA資料估算台灣黃芩與南台灣黃芩族群地理距離與族群分化指數之迴歸圖.............85
圖16. 利用粒線體DNA資料估算台灣黃芩與南台灣黃芩族群地理距離與族群分化指數之迴歸圖.............86
圖17. 利用細胞核核糖體DNA資料估算台灣黃芩與南台灣黃芩族群地理距離與族群分化指數之迴歸圖........87
圖18. 台灣黃芩與南台灣黃芩葉綠體DNA資料之基因型組成.......88
圖19. 台灣黃芩與南台灣黃芩粒線體DNA資料之基因型組成.......89


表 目 錄

表1. 台灣黃芩與南台灣黃芩採集明細........................13
表2. 聚合酶連鎖反應擴增cpDNA、mtDNA、nrDNA之引子序列......14
表3. 台灣黃芩與南台灣黃芩葉綠體DNA atpB-rbcL non-coding spacer之遺傳多型性分析.............90
表4. 台灣黃芩與南台灣黃芩粒線體DNA nad1 gene intron 2之遺傳多型性分析........................91
表5. 台灣黃芩與南台灣黃芩細胞核rDNA ITS之遺傳多型性分析....92
表6. 台灣黃芩與南台灣黃芩之cpDNA atpB-rbcL non-coding spacer、mtDNA nad1 intron 2、nrDNA ITS基因片段中,序列全長及核苷酸比值.........................26
表7. 台灣黃芩與南台灣黃芩葉綠體DNA atpB-rbcL non-coding spacer之族群間與種間核苷酸歧異度、淨歧異度分析..................93
表8. 台灣黃芩與南台灣黃芩粒線體DNA nad1 gene intron 2之族群間與種間核苷酸歧異度、淨歧異度分析...94
表9. 台灣黃芩與南台灣黃芩細胞核rDNA ITS之族群間與種間核苷酸歧異度、淨歧異度分析.................95
表10. 台灣黃芩與南台灣黃芩葉綠體DNA atpB-rbcL non-coding spacer之族群與種間遺傳分化指數及基因流傳值分析............96
表11. 台灣黃芩與南台灣黃芩粒線體DNA nad1 gene intron 2之族群與種間遺傳分化指數及基因流傳值分析.....................97
表12. 台灣黃芩與南台灣黃芩細胞核rDNA ITS之族群與種間遺傳分化指數及基因流傳值分析.................98
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