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研究生:吳淑華
研究生(外文):Wu Shu Wha
論文名稱:紅楠葉綠體DNA之親緣地理
論文名稱(外文):Phylogeography of Machilus thunbergii Based on Chloroplast DNA Polymorphism
指導教授:黃士穎
指導教授(外文):Shih-Ying Hwang
學位類別:碩士
校院名稱:中國文化大學
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:2004
畢業學年度:92
語文別:中文
論文頁數:63
中文關鍵詞:紅楠葉綠體DNA親緣地理遺傳歧異度
外文關鍵詞:Machilus thunbergiichloroplast DNAPhylogeographytrnF-trnLtrnM-trnVgenetic diversity
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本研究以葉綠體DNA trnF-trnL及trnM-trnV兩個基因間片段(intergenic spacer)的序列來分析紅楠(Machilus thunbergii)的族群遺傳歧異度及親緣地理。紅楠共採自27個族群,台灣26個族群,沖繩島1個族群,共計112個樣本。排序的結果總長度為1031 bp,共有16個多型性位點,其中3個為訊息位置,包含2個易位(transversion)及1個轉換(transition),13個為singleton,由TCS基因譜系分析共獲得10個獨特的單套型(haplotype),基因樹呈現星狀結構。共有99個樣本是屬於祖先單套型A,其中單套型A普遍存在各族群之中。在中央山脈的西側族群中,分佈在新竹的秀巒及台中的大雪山有最高的單套型歧異度(h),但族群內的單套型都屬於姊妹型(sister haplotype),也就是來自同一個祖先型;在中央山脈東側族群中,幾乎為祖先型A。藉由花粉化石紀錄和低核苷酸歧異度、中性假說Tajima’s D統計值呈現的顯著偏離零及族群變動分析呈現單一波峰結果的整合,推論紅楠的族群變動應該是處於歷經冰河期瓶頸事件後的後冰河族群成長階段。利用ARLEQUIN軟體測試葉綠體DNA和粒線體DNA之間的連鎖不平衡,結果顯示locus1 (trnF-trnL)和locus2(nad7/1-nad7/2r)之間有顯著的連鎖關係(P<0.02),和locus2(nad7/1-nad7/2r)之間僅有微弱的連鎖關係存在。
This study examined the spatial pattern of chloroplast DNA (cp DNA) variation in Machilus thunbergii in 112 samples from Taiwan (26 poplutions) and Okinawa (1 polpulation). By sequencing two cpDNA intergenic spacer fragments using universal primers (trnF-trnL and trnM-trnV). 16 polymorphic sites were found, of which three informative sites include two transitions, one transversion and 13 singletons were detected from the aligned 1031 base pair. We identified 10 unique haplotypes using the TCS program. Haplotype network depicted a “star-like” phylogeny. One ancestral haplotype A was found in 86 individuals and widely distributed among populations. Among the populations on the west of the CMR, Shiouluan and Dashiueshan had high haplotype diversity; each consisting of sister haplotypes all mutated from the ancestral haplotype. Among the populations on the east of the CMR ancestral haplotype dominates. One probable demographic history of Machilus thunbergii is the postglacial population growth of Machilus thunbergii after a glacial bottleneck event. This inference is supported by the combined results of fossil pollen record, low nucleotide diversity, significant Tajima’s D-value showed the negative values significantly different from zero and unimodal mismatch distribution. To test for an association between mitochondrial and chloroplast haplotypes, analysis were performed using the linkage disequilibrium exact test between cpDNA and mtDNA using ARLEQUIN. The result showed strong association in (1,2) (P<0.02) and weak linkage in (0,2).
目錄……………………………………………………………………………….Ⅰ
表目錄……………………………………………………………………….……Ⅱ
圖目錄……………………………………………………………………….……Ⅲ
摘要………………………………………………………………………………...1壹、前言……………………………………………………………………………3
貳、前人研究………………………………………………………………………5
參、材料與方法…………………………………………………………………..11
一、樣本的蒐集 ……………………………………………………………….11
二、樣本處理……………………………………………………………….….11
三、DNA萃取………………………………………………………………….11
四、DNA定量與稀釋………………………………………………………….12
五、聚合酶連鎖反應與定序…………………………………………….…….13
六、資料分析 ………………………………………………………………….15
(一) 序列分析……………………………………………………………..15
(二) TCS基因譜系分析………………………………………………….16
(三) 遺傳變異分析………………………………………………………..16
(1) 核苷酸歧異度(π) ………………………………………17
(2) 單套型歧異度(h)……………………...…………………..17
(3) 核苷酸分歧度(θ)…………………………………………17
(4) 中性假說測試(Tajima’s test) …………………………….18
(5) Fu and Li’s test …………………………………………...18
(四)族群次結構分析………………………………………………………..19
(五)族群分化指數(FST)及基因流傳值(Nm)分析………………………….21
(六) mismatch distribution分析………………………………….………..21
(七) 胞器DNA之連鎖不平衡(Linkage disequilibrium)分析……………22
肆、結果…………………………………………………………………………..23
伍、討論…………………………………………………………………….…….29
陸、參考文獻…………………………………………………………………….36
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