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研究生:許芯芯
研究生(外文):Hsin-Hsin Hsu
論文名稱:利用微衛星標幟與粒線體 DNA 探討臺灣地區大冠鷲之遺傳結構與多樣性
論文名稱(外文):Genetic Diversity and Genetic Structure of Crested Serpent Eagles in Taiwan Based on Microsatellite Markers and Mitochondrial DNA
指導教授:王佩華
指導教授(外文):Pei-Hwa Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:動物科學技術學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:91
中文關鍵詞:大冠鷲微衛星標幟粒線體DNA遺傳保育
外文關鍵詞:Crested Serpent Eaglemicrosatellite markermitochondrial DNAgenetic conservation
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猛禽主要面臨問題為棲地喪失、人類開發及活動影響,造成物種瀕臨絕種之危險。大冠鷲(Crested Serpent Eagle, Spilornis cheela hoya)在台灣屬於珍貴稀有保育類野生動物,利用遺傳分子標幟了解族群的遺傳歧異度(genetic diversity)及遺傳結構,有助於進行族群管理與遺傳保育。本試驗利用微衛星標幟與粒線體 DNA 控制區檢測臺灣地區 62 隻大冠鷲之遺傳變異,並利用其中已知來源樣本 42 隻樣本分析臺灣地區大冠鷲之遺傳結構。
利用 16 個白尾海雕及西班牙帝雕所開發的微衛星基因座,作為跨物種微衛星標幟的研究結果顯示,其應用於大冠鷲的效果不佳,僅有 11 個微衛星基因座具有多態性,平均異質度低(HE=0.38, HO=0.35),且經序列分析發現在大冠鷲所增幅的序列中,重複單位的數量大量減少,造成多態性降低。因此,本試驗遂以大冠鷲基因體篩選其適用之微衛星標幟,利用選擇性雜合法(selective hybridization)以(CA)n 與(GATA)n 重複單位作為探針,建構微衛星序列豐富之基因庫,並篩選出 24 個新的微衛星基因座。而平均每個基因座的交替基因數量(Na)為 2 至 8 個,且平均期望異質度與觀測異質度數值接近。以開發之微衛星基因座分析大冠鷲族群的遺傳結構結果顯示,臺灣各區域間的遺傳分化指數(FST)與祖先遺傳係數(RST)皆小於 0.05,顯示不同區域之間大冠鷲族群沒有遺傳分化的情況。此外,各個區域間的遺傳距離以東部與其他三個區域的遺傳距離數值大,推測係因高山所造成的輕微隔絕。
在大冠鷲粒線體 DNA 的研究方面,本試驗成功的選殖及定序大冠鷲粒線體基因體 18828bp,利用高變異之控制區(control region)序列設計引子對大冠鷲樣本進行增幅,序列分析結果顯示,在 62 個樣本中共可以發現 12 個單套型(haplotype),臺灣各區域間的遺傳分化指數與微衛星標幟分析結果符合,皆沒有明顯的分化情況。以單套型所繪製的最小親緣關係網狀圖(minimum spanning network)亦顯示臺灣各區域大冠鷲沒有明顯的分群現象。
本研究自大冠鷲基因體開發 24 個新的微衛星基因座,並將大冠鷲之粒線體基因體定序完成,不僅可作為遺傳監控的重要分子遺傳標幟,亦可作為相關研究之應用。此外,結合微衛星標幟與粒線體 DNA 控制區序列的多型性分析結果顯示,臺灣地區大冠鷲族群各區域間沒有明顯的分化情況,故可視為單一的管理單位,雖無嚴重的遺傳歧異度喪失,仍應持續進行遺傳監控以進行遺傳保育。


Raptor populations become fragmented and are often decreased due to human impact to nature environment. Crested Serpent Eagle (Spilornis cheela hoya) is endemic and protected animals in Taiwan. To manage and conserve this species, knowledge of the genetic diversity and genetic structure is critical. Microsatellite markers and mitochondrial DNA were analyzed for 62 Crested Serpent Eagles including 42 samples with known habitats in certain regions in Taiwan.
Cross-species microsatellite markers from White-tailed Sea Eagle and Spanish imperial eagle were used to amplify in this study. The results showed the markers were of limited use in Crested Serpent Eagles. There were only 11 microsatellite loci displayed polymorphisms in a total of 16 loci, and the mean heterozygosity was low (HE=0.38, HO=0.35). Moreover, the sequence in these loci showed decreased number of repeated motif in Crested Serpent Eagles. Therefore, we cloned 24 novel microsatellite markers from Crested Serpent Eagles using a selective hybridization method from a genome library enriched for (CA)n and (GATA)n repeat motifs. The allele numbers ranged from two to eight per locus, and the observed and expected heterozygosity were similar. Using these markers to analyze pairwise FST estimates, we found that limited Crested Serpent Eagles population differentiation in various regions of Taiwan (FST< 0.05, RST<0.05). Besides, the eagles in eastern region showed higher genetic distance from other regions. We also cloned and sequenced the mitochondrial genome (18828 bp) of the Crested Serpent Eagle, and designed a primer set to amplify a highly variable control region to decipher the genetic diversity of this species. We observed 12 haplotypes in 62 Crested Serpent Eagle samples. Similarly with the result from microsatellite markers, the pairwise FST estimates and haplotypes minimum spanning network revealed the eagles were limited differentiation and there was no obvious genetic distribution according to geographic segregation.
In conclusion, we developed 24 microsatellite markers from Crested Serpent Eagles and sequenced its mitochondrial genome. These tools could be very useful for genetic monitoring and other application in the future. Moreover, the microsatellite markers and mitochondrial DNA data showed that Crested Serpent Eagles in Taiwan have limited genetic differentiation and there are sufficient genetic diversity. Therefore the Crested Serpent Eagle in Taiwan should be recognized as a single management unit for conservation programs.


謝誌 Ⅰ
目錄 Ⅲ
圖次 Ⅴ
表次 Ⅵ
附錄 Ⅶ
中文摘要 Ⅷ
英文摘要 Ⅹ
第一章 文獻檢討 1
壹、遺傳保育 1
一、瀕臨絕種鳥類 1
二、遺傳保育研究 2
貳、大冠鷲之生物學研究 5
參、遺傳保育 DNA 分子遺傳標幟 7
一、粒線體 DNA 8
二、核內 DNA 13
三、利用 DNA 分子遺傳標幟進行猛禽遺傳保育之應用 22
肆、研究目的 25
第二章 材料與方法 26
試驗一、跨物種微衛星標幟 26
試驗二、大冠鷲微衛星標幟開發與應用 31
試驗三、大冠鷲粒線體基因體定序與控制區域序列分析 38
第三章 結果 42
試驗一、跨物種微衛星標幟 42
ㄧ、微衛星基因座與遺傳變異 42
二、微衛星標幟序列分析 42
試驗二、大冠鷲微衛星標幟開發與應用 47
一、大冠鷲微衛星標誌篩選 47
二、以微衛星標幟分析大冠鷲微基因歧異度 47
三、以微衛星標幟分析大冠鷲微基因結構 48
試驗三、大冠鷲粒線體基因體定序與控制區域序列分析 55
一、大冠鷲粒線體 DNA 基因體全序列定序 55
二、控制區序列分析 55
第四章 討論 61
一、 跨物種微衛星標幟 61
二、大冠鷲微衛星標幟篩選 61
三、粒線體 DNA 序列 63
四、臺灣地區大冠鷲遺傳歧異度 63
五、臺灣地區大冠鷲遺傳結構 64
六、保育管理政策評估 65
第五章 結論 67
參考文獻 68
附錄 80


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