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研究生:孫于婷
研究生(外文):Yu-Ting Sun
論文名稱:臺灣梅花鹿遺傳多樣性與族群遺傳結構研究
論文名稱(外文):The genetic diversity and population genetic structure of Formosan sika deer
指導教授:朱有田朱有田引用關係姜延年姜延年引用關係
口試委員:王穎林思民顏聖紘
口試日期:2014-07-28
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:2014
畢業學年度:102
語文別:中文
論文頁數:79
中文關鍵詞:臺灣梅花鹿微衛星標記族群遺傳結構
外文關鍵詞:Formosan sika deermicrosatellite markerpopularion genetic structure
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摘要
臺灣梅花鹿 (Cervus nippon taiouanus) 為臺灣特有亞種,約於西元1969年在野外絕跡。臺灣梅花鹿復育計畫在西元1984年展開,由臺北市立動物園圓山舊址引入5雄17雌梅花鹿於墾丁國家公園進行復育,而後又加入少數民間鹿群。現今野外族群已成長到1,500隻左右。原始復育族群個體數目少、非源自野外且來源分歧等因素,因此族群遺傳結構、遺傳多樣性與外來鹿科動物之基因滲入問題需要被研究。
本研究以跨物種微衛星DNA標記為分析工具。自牛科微衛星標記篩選11組在臺灣梅花鹿具微衛星特徵與多形性之標記,分析墾丁國家公園90頭臺灣梅花鹿、金門畜試所36頭、大坵島39頭、壽山動物園8 頭及民間飼養梅花鹿23頭進行遺傳多樣性檢測。同時,分析族群內遺傳多樣性及族群間分化指數,並以遺傳距離建構族群親緣關係樹。另外,本研究整合粒線體母系遺傳資訊與Y染色體Zfy (zinc-finger gene in Y-chromosome) 鋅指基因父系遺傳資訊,進行物種鑑定並和11組微衛星資料做比對,以探討過去基因滲入鹿群的現象。
檢測11組微衛星標記在196頭臺灣梅花鹿整體之個體鑑別率為 (P(ID))為2.06×10-6,多態性訊息含量(PIC)為0.439,顯示所選微衛星標記適合進行臺灣梅花鹿遺傳多樣性分析。在遺傳多樣性分析結果,顯示墾丁梅花鹿族群較臺灣其他族群含有較高遺傳多形性與異型合子歧異度。墾丁國家公園梅花鹿復育族群與金門畜試所、馬祖大坵梅花鹿族群三者在親緣遺傳關係上較近,與圈養族群親緣較遠。另外,兩民間飼養鹿群間分化指數與遺傳距離小,顯示互有基因交流。在基因滲入情況研究上,本研究亦建立臺灣梅花鹿與紅鹿或其他外來鹿科動物之母系Cyt b與Y染色體上父系Zfy序列資料庫做為初步物種鑑定依據,並配合微衛星標記基因型分析,結果顯示民間鹿場有較明顯的紅鹿基因滲入,墾丁國家公園梅花鹿復育族群與金門畜試所、馬祖大坵梅花鹿族群沒有顯著紅鹿基因滲入。
綜上所述,本研究所篩選11組跨物種微衛星標記適合研究臺灣梅花鹿遺傳多樣性,輔以Cyt b與Zfy序列更可分析外來鹿科動物基因滲入情況。墾丁國家公園梅花鹿族群、金門畜試所及大坵島族群紅鹿基因滲入比例極低,且與民間飼養梅花鹿已有明顯遺傳分化現象,彰顯過去臺灣梅花鹿復育重要性。本研究提供臺灣臺灣梅花鹿遺傳結構、分化與多樣性基本資訊。提供族群遺傳管理與防範外來鹿科動物基因滲入、遺傳監控與法醫學鑑定方法。


ABSTRACT
Formosan sika deer (C. n. taiouanus) is endemic to Taiwan, and was widely distributed in the woodlands under 300 m elevation. The wild Formosan sika deer was extinct in 1960, while some individuals were conserved in Taipei Zoo and deer farm. The Recovering Program of Formosan sika deer started in 1986, and Twenty-two individuals were chosen from Taipei zoo and conserved in the wild of Kenting National Park. The population size of wild deer in Kenting National Park has increased to about 1,500 nowadays (2010). However, the genetic structure of the wild Formosan sika deer is not clear. The research aim to select applicable microsatellite markers for Formosan sika deer; to investigate the genetic diversity and genetic structure of Formosan sika deer conserved population for population management; to analyze the introgression from red deer to sika deer populations.
A total 196 sika deer samples form 6 sites were collected (90 from Kenting National Park, 36 from Kinmen Livestock Research Institute, 39 from Ta Chiu islet, 8 from Shou Shan Zoo, 10 form Taitung deer farm and 13 from Tainan deer farm) for population study. The cross-species microsatellite markers were selected form 22 bovine and ovine markers. The markers with short tandem repeat sequences were examined cloning to separate sequences and sequencing. The genetic diversity and genetic structure of Formosan sika deer was examined by 11microsatellite markers. Identification of species is conducted by Zfy gene and cytochrome b for investigating cultivated deer populations.
The selected 11 crossed-species markers showed medium level of polymorphic information content in whole Formosan sika deer population. And the probability of identity (P(ID)) is less than 0.0001. These results reveal The selected 11 crossed-species markers are polymorphic and achieve a very low probability of finding two random individuals with identical genotypes. The average polymorphic information is 0.439 in Kenting National Park population (KNP). The average expected heterozygosity (He) and observed heterozygosity (Ho) were both 0.512. It showed KNP population maintained more alleles and heterozygosity than other conservation populations. The result of Neighbor-Joining tree showed KNP, Kinmen and Ta Chiu islet population can cluster together. It agrees with the process of introduced founder populations. Utilizing Zfy gene and Cytochrome b gene can acquire maternal and paternal genetic information. The genetic structure showed red deer population has different pattern with sika deer population. And only a few individuals in deer farm showed introgression.
In conclusion, this study can provide the applicable method to investigate population genetic structure of Formosan sika deer with microsatellite markers. The information of genectic diversity in Kenting National Park population can be refer as population management. With microsatellite, Zfy and Cytochrome b gene, the futher study of introgression from red deer to sika deer population can be implemented.


目錄
摘要……………………………………………………………………………………Ⅵ
Abstract………………………………………………….…………………………….Ⅷ
前言 ……………………………………………………………………………………1
壹、文獻探討…………………………………………………………………………..2
一、臺灣梅花鹿之分類地位及與歷史介紹……………………………………………2
(一)臺灣梅花鹿形態特徵與習性簡介…………………………………………..2
(二)臺灣梅花鹿之野外滅絕與復育……………………………………………..2
(三)現今臺灣梅花鹿族群概況…………………………………………………..3
(四)臺灣養鹿事業與飼養鹿群介紹………………………………………….….4
二、分子遺傳標記之應用……………………………………………………………..5
(一)分子遺傳標記…………………………………………………………….….6
(二)微衛星標記(microsatellite marker)………………………………………6
(三)Y染色體鋅指基因(Y-linked zinc finger gene, Zfy)………….…………7
(四)粒線體細胞色素b(cytochrome b)基因……………………………….…7
三、分子遺傳標記於鹿科動物遺傳研究之應用………………………………….….7
(一)跨物種微衛星標記之應用…………………………………………….….…7
(二)以微衛星標記分析基因滲入……………………………………………..…8
(三)以Zfy基因做親緣關係之分析………………………………………….…..8
四、研究目的……………………………………………..……………………………9
貳、材料方法……………………………………………..……..……………………10
一、收集原生梅花鹿及非原生紅鹿遺傳樣本…………..……………….………….10
(一)鹿隻血液與組織樣本採集與整…………………………….…………….…10
(二)細胞核基因組DNA(genomic DNA)之萃取……………………………11
(三)分析紅鹿之細胞色素b與Y染色體鋅指基因序列,以鑑定鹿種………….12
二、跨物種微衛星標記之篩選……………………………………………………… 14
(一)引子挑選與設計………….…………………………………………………14
(二)微衛星序列選殖與定序……………………………………………………14
三、 微衛星基因座聚合&;#37238;連鎖反應與毛細管電泳………………………………....15
(一)增幅樣本之微衛星序列……………………………………………………15
(二)樣本之基因型資料收集…………………………………………………….16
四、應用微衛星標記多型性進行臺灣梅花鹿族群遺傳分析………………………16
(一)分析11組微衛星基因座多型性及個體鑑別率…………………………...16
(二)分析臺灣梅花鹿族群之遺傳多樣性………………………………………17
(三)估算臺灣梅花鹿族群之F-統計值(FST, FIS)………………………….19
(四)計算臺灣梅花鹿族群間之遺傳距離並建構類緣關係樹圖………………19
(五) 估算臺灣梅花鹿族群遺傳結構……………………..……………………20
五、應用微衛星標記多型性分析臺灣梅花鹿與紅鹿之差異………………………20
(一)建立紅鹿微衛星基因型資料庫並估算基因滲入程度……………………20
參、結果………………………………………………………………………………22
一、篩選適合作為分析臺灣梅花鹿之跨物種微衛星標記…………………………22
(一)收集臺灣梅花鹿微衛星序列………………………………………………22
(二)臺灣梅花鹿微衛星標記對偶基因型之毛細管電泳標準波形……………22
二、應用微衛星標記多型性進行臺灣梅花鹿族群遺傳分析………………………23
(一)分析11組微衛星基因座多型性及個體鑑別率…………………………..23
三、臺灣梅花鹿族群遺傳分析………………………………………………………23
(一)臺灣梅花鹿各族群遺傳多樣性分析與標記使用之限制…………………23
(二)臺灣梅花鹿族群間族群分化與遺傳距離…………………………………26
(三)臺灣梅花鹿族群遺傳結構…………………………………………………27
四、紅鹿基因滲入臺灣梅花鹿之分析………………………………………………28
(一)紅鹿及雜交鹿隻物種鑑定…………………………………………………28
(二)紅鹿微衛星對偶基因資料收集……………………………………………28
(三)以遺傳結構檢測紅鹿基因滲入梅花鹿族群………………………………29
肆、討論………………………………………………………………………………30
一、探討跨物種微衛星標記於臺灣梅花鹿之應用…………………………………30
(一)臺灣梅花鹿微衛星標記之篩選方式………………………………………30
(二)11組微衛星標記之限制與鑑別率…………………………………………31
二、臺灣梅花鹿族群之遺傳多樣性…………………………………………………31
(一)臺灣梅花鹿遺傳多樣性……………………………………………………31
(二)臺灣梅花鹿族群內近親交配係數…………………………………………32
(三)臺灣梅花鹿族群分化與遺傳距離…………………………………………33
(四)臺灣梅花鹿遺傳結構………………………………………………………33
三、外來紅鹿基因滲入………………………………………………………………34
(一)遺傳樣本限制與代表性……………………………………………………34
(二)以Zfy基因做親緣關係之分析……………………………………………35
(三)鹿隻遺傳結構………………………………………………………………35
五、結論………………………………………………………………………………36
陸、圖與表……………………………………………………………………………37
柒、參考文獻…………………………………………………………………………55
附錄……………………………….……………….…………………………………..60


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