姜延年臺灣大學:動物科學技術學研究所吳宗育Wu, Chung-YuChung-YuWu2010-05-112018-06-292010-05-112018-06-292008U0001-2907200802152500http://ntur.lib.ntu.edu.tw//handle/246246/182039桃園豬於1877年自廣東地區被引進，並成為臺灣早期主要飼養之豬種。為了改進性能，於1896年後，盤克夏、約克夏、漢布夏、藍瑞斯、杜洛克等外來豬種陸續被引入臺灣。由於純種桃園豬的經濟性狀較外來豬種差，造成飼養意願降低，使得其數量遽減。為了進行保種，桃園豬在1974年後被隔離飼養於臺灣省畜產試驗所。蘭嶼豬為蘭嶼島上之在地豬種，並於1975年與1980年分別自蘭嶼島引種至國立臺灣大學與臺東種畜繁殖場，隔離飼養作為發展實驗用豬種之研究。由引種時間推論，外來豬種之基因有滲入桃園豬與蘭嶼豬之可能。然而桃園豬與蘭嶼豬這兩個保種族群內部的遺傳結構，迄今仍未知。瞭解保種族群的遺傳多樣性，將有助於建立往後保種工作之方針，故有其重要性。研究乃根據粒線體序列之多型性，探討下列主旨：(1) 計算桃園豬以及蘭嶼豬保種族群內部粒線體序列的遺傳變異程度；(2) 確認外來豬種的粒線體遺傳資訊是否曾經滲入保種族群當中；(3) 分析桃園豬、蘭嶼豬保種族群與全世界豬種間之親緣關係。收集藍瑞斯、約克夏、杜洛克、盤克夏、梅山豬、保種桃園豬、保種蘭嶼豬共129隻的粒線體樣本，並以聚合酶鏈反應增幅其控制區域與細胞色素b片段，再進行純化與定序。另外43筆來自NCBI資料庫的歐亞豬種控制區域序列，亦被收集並納入計算。根據基因型歧異度 (h) 與核苷酸歧異度 (π)，分析桃園豬與蘭嶼豬保種族群內部的遺傳歧異度。另根據Hasegawa-Kishino-Yano 85 (HKY85)之演化模式，將粒線體控制區域與細胞色素b序列變異之多型性資訊，轉換成為遺傳距離，最後利用兩兩序列間的遺傳距離，建構Maximum Likelihood (ML) 樹狀圖。園豬保種族群的控制區域與細胞色素b序列，皆僅出現一種基因型，並且由 h 值 (0.000) 與π值 (0.000%) 顯示出低度的歧異度；蘭嶼豬保種族群的控制區域與細胞色素b序列，皆可被分為兩種基因型（第一型與第二型），並且由 h 值 (0.384) 與 π值 (0.510%) 顯示其歧異度的嚴重流失。在控制區域與細胞色素b序列的親緣關係分析中，第一型蘭嶼豬序列與其他豬隻序列分離，並形成一獨特類群；第二型蘭嶼豬則與約克夏、盤克夏、梅山豬以及桃園豬歸類於同一類群。在包含184頭歐亞豬隻控制區域序列的ML樹狀圖中，第一型蘭嶼豬序列仍然與其他歐亞豬隻序列分離，形成一獨特類群；第二型蘭嶼豬與桃園豬序列則同樣被歸類於亞洲豬種序列。合上述結果，桃園豬與蘭嶼豬保種族群皆出現低度的遺傳歧異度，顯示瓶頸效應或是創始者效應在這些族群中，有曾經發生過之可能。桃園豬保種族群序列被歸類於亞洲類群，顯示歐洲豬種的母方遺傳資訊，未滲入受測桃園豬保種族群當中。第一型蘭嶼豬序列與其他歐亞豬隻序列分離，並形成一獨特類群，可能是由於第一型蘭嶼豬具有與其他歐亞豬種不同起源之母方遺傳資訊。第二型蘭嶼豬序列被歸類於亞洲主類群中，可能是由於亞洲主類群中的豬種，其母方遺傳資訊曾經滲入蘭嶼豬當中，所造成之結果。Taoyuan pigs were introduced from Guangdong area in 1877 and were a major breed in early Taiwan. For improving their performance, Berkshire pigs were introduced into Taiwan by Japanese in 1896. Then according to their poor economic efficiency and lower willing of rearing, the number of pure Taoyuan pigs decreased dramatically after the exotic pig breeds (Yorkshire, Hampshire, Landrace, and Duroc) were introduced. For animal conservation, Taoyuan pigs were reared isolately in Taiwan Livestock Research Institute after 1974. Lanyu pigs, an indigenous breed in Lanyu Islet were transferred to National Taiwan University and Taitung Animal Propagation Station for developing laboratory swine in 1975 and 1980, respectively. Two herds of Lanyu pigs were reared in isolation and performed natural mating until 2004 and 1991. The genes of exotic pig breeds might introgress into Taoyuan and Lanyu pigs after exotic breeds introduced into Taiwan. The genetic diversity within conserved Taoyuan and Lanyu herds and whether the genetic introgression had previously occurred were currently unknown, so it is important identifying the genetic diversity of conserved herds for the orientation of conservation. he aims of the study were to investigate: (1) the genetic variation within conserved herds of Taoyuan and Lanyu pigs. (2) Whether the maternal genetic introgression from exotic pig breeds into the conserved herds (3) the phylogenetic relationships of two conserved breeds to worldwide pig breeds. The polymorphisms of mitochondrial sequences were applied for those studies. Mitochondrial DNAs of 129 individuals from Landrace, Yorkshire, Duroc, Berkshire, Meishan, and conserved herds of Taoyuan and Lanyu pigs were obtained. Their control region and cytochrome b fragments were amplified by polymerase chain reaction then sequenced. Genetic diversities within conserved herd sequences of Taoyuan and Lanyu pigs were examined based on the relationships of haplotype diversity (h) and nucleotide diversity (π). The Hasegawa-Kishino-Yano 85 (HKY85) substitution model was used to obtain the genetic distances based on the polymorphisms of substitution sites of control region and cytochrome b sequences. The Maximum Likelihood (ML) tree of different sequences was constructed according to the pairwise genetic distances. To determine the phylogenetic relationship among Taoyuan, Lanyu, and other Eurasian pig breeds, 55 control region sequences of Eurasian pig breed obtained from NCBI database were included.he control region and cytochrome b sequences of conserved Taoyuan only remained one haplotype, and shown absence of diversity by low h (0.000) and lowπ(0.000%) value; conserved Lanyu sequences were divided into two haplotypes (type I and type II), and shown severe loss of diversity by low h (0.384) and highπ(0.510%) value. In the phylogenetic relationship of control region and cytochrome b sequences, type I Lanyu formed a unique clade different from all other pigs while type II Lanyu sequence was clustered with Yorkshire, Berkshire, Meishan, and Taoyuan. In ML tree containing 184 control region sequences of Eurasian pigs, type I Lanyu sequence still formed a unique clade different from Asian and European pig sequences, while type II Lanyu and Taoyuan sequences were clustered within the Asian clade.n conclusion, low genetic diversity observed both in conserved Taoyuan and Lanyu pigs indicates recent population bottleneck or founder event might happen in these herds. Taoyuan sequence was clustered in Asian clade indicate that maternal lineage introgression from European clade pigs wasn’t found. Type I Lanyu sequence formed a unique clade different from other Asian and European sequences suggesting that the origin of Lanyu pigs have maternal lineage distinct from other Asian and European pigs. Type II Lanyu sequence was clustered within the Asian clade, it might be the result of maternal lineage introgression from Asian clade pigs into Lanyu pigs.口試委員會審定書 i謝 ii文摘要 iv文摘要 vi言 viii、文獻檢討 1一章 臺灣本地與外來豬隻品種之歷史介紹 1一節 歐美品種 1二節 亞洲品種 4二章 物種分類之依據 7一節 形態標記 7二節 細胞遺傳標記 7三節 免疫遺傳標記 8四節 生化標記 8五節 分子遺傳標記 9三章 分子遺傳標記的多型性分析 11一節 限制片段長度多型性 11二節 隨機增幅多型性DNA 11三節 增幅片段長度多型性 12四節 簡單序列長度多型性 12五節 單一核苷酸多型性 13四章 遺傳多型性在物種親緣關係之應用 14一節 族群遺傳結構分析 14二節 遺傳距離計算 15三節 親緣關係圖繪製 15四節 演化分歧時間估算 16、材料方法 17一) 血液樣本收集 17二) 血樣採集 17三) 粒線體DNA萃取 18四) 聚合酶鏈反應 18五) DNA定序 19六) 序列收集與整理 20七) 統計軟體分析 20、序列排序比對 20、族群結構分析與演化分歧時間估算 21、遺傳距離矩陣計算 21、親緣關係圖繪製 22、結果 23一) 保種群桃園豬之族群內遺傳歧異度分析 23二) 保種群蘭嶼豬之族群內遺傳歧異度分析 23三) 保種群桃園豬、蘭嶼豬與其他外來豬隻族群間親緣關係分析 24四) 保種群蘭嶼豬、蘭嶼島現存豬隻與其他外來豬隻族群間親緣關係分析 24五) 保種群桃園豬、蘭嶼豬與其他歐亞豬隻族群間核苷酸多型性分析 25六) 保種群桃園豬、蘭嶼豬與其他歐亞豬隻族群間親緣關係分析 26七) 演化分岐時間之估算 27、討論 28一) 保種群桃園豬族群之遺傳多樣性 28二) 保種群蘭嶼豬族群之遺傳多樣性 28三) 保種群桃園豬、蘭嶼豬與其他外來豬隻族群間粒線體基因滲入之情形 29四) 蘭嶼島現存豬隻的外來豬隻粒線體基因滲入情形 29五) 保種群桃園豬、蘭嶼豬與其他歐亞豬隻族群間親緣關係 30六) 蘭嶼豬序列之可能形成過程 31七) 歷史上蘭嶼豬序列之分布情形 32、參考文獻 33、表與圖 431. 第一型蘭嶼豬、第二型蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克、盤克夏族群間控制區域與細胞色素b合併序列之遺傳距離矩陣 431. 國立臺灣大學與臺東種畜繁殖場保種群蘭嶼豬在控制區域與細胞色素b序列的核苷酸變異位置 442. 國立臺灣大學與臺東種畜繁殖場保種群蘭嶼豬在控制區域序列的重複片段 453. 第一型蘭嶼豬、第二型蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克、盤克夏族群間控制區域與細胞色素b合併序列之親緣關係樹狀圖 464. 保種群蘭嶼豬、蘭嶼島現存豬隻，以及外來豬種間控制區域序列之親緣關係樹狀圖 475. 歐亞豬種於細胞色素b序列之核苷酸變異位置 496. 歐亞豬種於控制區域序列之核苷酸變異位置 507. 歐亞豬種間細胞色素b序列之親緣關係樹狀圖 528. 歐亞豬種間控制區域序列之親緣關係樹狀圖 54、附錄 561. 對於族群內單套型歧異度與核苷酸歧異度之間的關係，在各種不同狀態下之解釋 562. 自NCBI資料庫中，取得控制區域序列之豬隻編號、名稱、譯名以及棲息地 573. 自NCBI資料庫中，取得細胞色素b序列之豬隻編號、名稱、譯名與棲息地 591. 澳洲、紐西蘭野化家豬與歐亞家豬、野豬於控制區域序列之核苷酸變異位置 602. 第一型蘭嶼豬粒線體核苷酸全長序列 623. 第二型蘭嶼豬粒線體核苷酸全長序列 68、作者小傳 74application/pdf1164671 bytesapplication/pdfen-US桃園豬蘭嶼豬粒線體序列Taoyuan pigLanyu pigmitochondrial sequencethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182039/1/ntu-97-R95626020-1.pdf