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研究生:洪茂森
研究生(外文):Mao-Sen Hung
論文名稱:以4-coumarateCoAligase基因進行台灣冷杉族群親緣地理關係研究
論文名稱(外文):Phylogeography of Abies kawakamii (Hayata) Ito based on 4-coumarate CoA ligase gene.
指導教授:何國傑何國傑引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:59
中文關鍵詞:台灣冷杉親緣地理
外文關鍵詞:phylogeographyAbies kawakamii
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本研究以核內基因4- coumarate CoA: ligase (4CL)的序列片段 (約1174 bp),分析台灣冷杉 (Abies kawakamii (Hayata) Ito)的族群結構及親緣地理關係。台灣冷杉樣本採自八個族群,分別位於大霸尖山 (8株)、雪山 (8株)、合歡山 (9株)、奇萊山 (8株)、北二段 (6株)、玉山 (8株)、關山 (8株)以及南湖大山 (8株),以每個單株的基因體為模板進行PCR反應後,再將產物進行選殖,每個單株取兩個選殖株進行定序,共分析了135個序列。對這些序列分析發現可分為兩群,顯示其中可能包含兩個獨立基因,命名為4CL1及4CL2。我們選取其中4CL1基因共88個序列做後續研究,序列中包含五個exon,四個intron。Intron總長度約為380 bp,其中包含65個多型性位點,其中60個來自點突變 (point mutation),5個來自插入或刪除 (indel),共有71個基因型,大部分混合分布於各族群當中。若以個別的intron進行分析,則可發現四個intron所獲得基因樹呈現一致的規律。大多數的序列歸屬於原始型及衍生型兩種基因型,其餘則為末端型。各族群的基因型除只有原始型、衍生型外,均為非常unique的末端型為主。這些族群正往高海拔退縮,同時歷經瓶頸效應與基因漂變力量的作用,導致混合的基因型而很可能是避難族群的特色。而根據族群間FST/KST與Dxy所得數值做平均分別找到雪山、玉山與雪山、北二段、奇萊為最高值,為分化最強的區域,可能是主要的避難族群所在。
根據4CL1 DNA序列或是種子形態,Abies homolepis或Abies mariesii可能皆非台灣冷杉的祖先。
我們針對4CL1基因座數目進行研究,發現台灣冷杉種子內的配子體(haploid)中至少包含16個以上的4CL基因座。我們選用的基因4CL1可能有6個以上的基因座,而且序列非常相似且序列間只有singleton的差異。
Population genetic structure and phylogeographical relationships of Abies kawakamii (Hayata) Ito in Taiwan were studied by analyzing the DNA sequence of 4- coumarate CoA: ligase (4CL, 1174 bp). Samples were collected from eight alpine populations: Kuanshan, Yushan, Chilaishan, Hohuanshan, Nanhutashan, Tapachienshan, Tahsuenshan, and Wumingshan. Two clones of 4CL gene from each individual were sequenced. From a total of 135 DNA sequences of 68 individuals, 4CL can be separated into two gene family: 4CL1 and 4CL2. One of the gene, 4CL1 containing 88 sequences was further studied. The DNA fragment of 4CL1 consisted of five exons and four introns. The total length of intron was 380 bp, and contained 65 polymorphic sites: 60 point mutations and five insertions, giving a total of 71 haplotypes. The genealogical trees generated from these four introns showed similar pattern. Most of the sequences of each intron belonged to ancestral haplotype and derived haplotype, and many unique terminal haplotypes. Populations shared ancestral haplotype, derived haplotype, and the unique terminal haplotypes. These populations were retreating to higher altitude, experiencing an effect of bottleneck and genetic drift resulting in the composition of haplotype mentioned above that could characterize the refuge populations. Based on the average values of parameters of population differentiation, FST and KST, and population divergence DXY, we found that Tahsuenshan, Yushan, Wumingshan, and Chilaishan had the highest values indicating the possible major region of refugia for Abies kawakamii.

According to 4CL1 DNA sequence and the morphology of seed, neither A. homolepis nor A. mariesii of Japan is ancestral to A. kawakamii. In terms of number of loci of 4CL1 gene, we found six loci among 16 in the gametophyte. These 4CL1 DNA sequences were very similar and only exhibited single nucleotide polymorphism.
目錄
摘要…………………………………………………………………………………….I
目錄…………………………………………………………………………………..III
圖表目錄……………………………………………………………………………...V
壹、前言……………………………………………………………………….……….1
一、 台灣冷杉的生態資料……………………………………………….……..1
二、 親緣地理學………………………………………………………….…......1
三、 中性理論…………………………………………………………………...4
四、 基因漂變……………………………………………………………….......5
五、 基因譜系分析與溯祖理論………………………………………………...6
六、 台灣冷杉的遺傳性質……………………………………………………...9
貳、材料與方法……………………………………………………………………...10
一.樣本的採集………………………………………………………………....10
二.實驗方法……………………………………………………………………10
(一) 植株總DNA的萃取……………………………………………….…10
(二) 種子內總DNA的抽取…………………………………………….…11
(三) 總RNA的萃取……………………………………………………….13
(四) 聚合酶鏈鎖反應…………………………………………………..…14
(五) Gel extraction and TA-cloning…………………………………..……15
(六) 質體萃取……………………………………………………..………15
(七) Genomic walking…………………………………………………..….16
(八) Rapid amplification of cDNA ends (RACE)…………………….……18
三.資料分析…………………………………………………………….……...21
(一) 序列分析……………………………………………………….…….21
(二) 遺傳變異分析………………………………………………………..21
(1) 核苷酸歧異度 (π)及單套型歧異度 ( h ) ……………….…….22
(2) 中性假說測試……………………………………………….….22
(三) 族群次結構分析..................................................................................24
叁、結果………………………………………………………………………….….26
一、台灣冷杉核內基因4 coumarate-CoA ligase (4CL)的釣取………….…..26
二、台灣冷杉4 coumarate-CoA ligase 5’端與3’端cDNA序列的獲取….…26
三、不同台灣冷杉4CL DNA序列分析……………………………………...27
四、配子體內4CL之基因家族……………………………………………..…28
五、族群遺傳變異分析………………………………………………………..28
六、中性假說測試……………………………………………………………..29
七、台灣冷杉的親緣關係樹………………………………………….………..29
八、Mismatch pairwise distribution…………………………………………….29
肆、討論………………………………………………………………………………30
伍、參考文獻…………………………………………………………………………35

圖表目錄
圖一、台灣冷杉採集樣區分布圖……………………………………………………40
圖二、genomic walking 示意圖……………………………………………………..41
圖三、利用genomic walking所得4CL基因片段………………………………….42
圖四、4CL mRNA序列全長…………………………………………………………43
圖五、Intron I , II , III , IV遺傳譜系分析…………………………………………..44
圖六、各族群中原始型、衍生型、末端型三類基因型於intron III基因樹的分      布……………………………………………………………………………..48
圖七、族群關係圖……………………………………………………………………50
圖八、台灣冷杉親緣關係樹…………………………………………………………51
圖九、mismatch pairwise distribution分析…………………………………………..52
圖十、台灣冷杉與日本種冷杉A. homolepis, A. mariesii種子的外觀形態………53

表一、台灣冷杉採集地點列表………………………………………………………54
表二、引子名稱及序列………………………………………………………………55
表三、台灣冷杉各族群之核苷酸歧異度、單套型歧異度以及中性假說測試……56
表四、各區域核酸變異位置數量統計表……………………………………………57
表五、族群分化指數…………………………………………………………………58
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