姜延年臺灣大學：動物科學技術學研究所張偉華Chang, Wei-HuaWei-HuaChang2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/56645蘭嶼豬為蘭嶼島上之本地豬種，由於其體型小而被歸類為迷你豬，在1975年與1980年，為了發展醫學及生物實驗用豬種，而分別為臺灣大學畜牧系及農委會臺東種畜繁殖場由蘭嶼島引種至臺灣進行保種。近30年來，臺灣的保種蘭嶼豬隔離成臺灣大學及臺東種畜繁殖場兩個豬群並採自然配種方式進行飼養。由於並未保留完整的配種記錄，所以保種蘭嶼豬個體間之遺傳關係尚待釐清，作為族群管理之參考。此外自1877年起包括桃園豬、梅山豬、藍瑞斯、漢布夏、杜洛克、盤克夏與約克夏陸續引進臺灣，保種蘭嶼豬與這些豬種間也存在基因交流之可能性。 為了釐清以上之問題。此次研究利用微衛星標記之多型性，探討蘭嶼豬族群內之遺傳多型性及與臺灣外來豬種之類緣關係，並期望作為日後族群管理之參考。自農委會畜試所種原庫、臺東種畜繁殖場、高雄種畜繁殖場與國立臺灣大學取得桃園豬、梅山豬、藍瑞斯、蘭嶼豬、杜洛克、與約克夏7個豬種共242頭之血液檢體。並抽取其Genomic DNA後，經螢光標定之引子對豬15對染色體上之19個微衛星標記以PCR方式進行增幅。再以毛細管電泳之方式測定增幅後之片段長度，並根據其多型性估算此7豬種間之遺傳距離。結果顯示蘭嶼豬與梅山豬、盤克夏與約克夏間有較近之遺傳距離。而以遺傳距離建構7豬種間之Neighbor-Joining tree（NJ tree）並經1000次再取樣分析，結果顯示7個豬種間形成兩個主要且穩定之分支（bootstrap value = 99.6），其一包含藍瑞斯、杜洛克、盤克夏與約克夏4個歐美豬種。另一則由桃園豬、梅山豬與蘭嶼豬3個亞洲豬種所構成，且蘭嶼豬於此分支中又另形成一獨立分支（bootstrap value = 73.6）。此外根據7豬種242個個體間之遺傳距離所建構之NJ tree顯示，保種蘭嶼豬44個個體構成一獨立之分支。且未混入其他豬種。綜合以上族群與個體間之NJ tree結果顯示蘭嶼豬與上述6個臺灣地區外來豬種基因交流之可能性低，而為一獨特之豬種。 此次研究中對蘭嶼豬保種族群，以19組微衛星基因座之基因頻率進行哈溫平衡檢定，共有15組微衛星基因座顯著偏離哈溫平衡（P<0.05）。且FIS於所有的微衛星基因座上均為正值。此結果顯示蘭嶼豬保種族群可能遭受近親配種或Walhund effect。所以為了避免近親衰退或為維持保種蘭嶼豬族群內之遺傳多型性，可利用微衛星標記多型性估算其族群結構，以釐清各個個體間之遺傳關係。結果顯示保種蘭嶼豬可分成4個次族群，其中臺灣大學蘭嶼豬獨立形成一次族群，每一個體被分在此次族群中之機率均高於80％。而臺東種畜繁殖場之個體另外形成3個次族群。此結果顯示臺灣大學蘭嶼豬族群於此19個微衛星基因座上已形成與臺東種畜繁殖場不同之對偶基因型頻率。而兩族群間之遺傳距離亦同時支持此推論。 綜合以上之結果，推論保種蘭嶼豬與以上6個臺灣外來豬種間發生基因交流之可能性低為一獨特之族群，但此族群可能因近親配種致其基因多型性流失。而此次試驗根據微衛星標記多型性所估算之族群結構與其個體間遺傳距離矩陣，將可協助育種者釐清族群內之遺傳關係，而作為日後育成近親選育蘭嶼豬實驗品系或制訂保種計畫之參考。The Lanyu pig is an indigenous pig breed from Lanyu Islet and is classified as a miniature pig breed. For the purpose of developing laboratory swine, Lanyu pigs were introduced into Taiwan by the Department of Animal Husbandry, National Taiwan University and Taitung Animal Propagation Station in 1975 and 1980, respectively. These pigs were reared in isolation as two herds and performed random mating until 2004. The genetic diversity within all the conserved Lanyu pigs remained unknown. Many exotic pigs breeds including Taoyuan, Meishan, Landrace, Duroc and Yorkshire breeds were introduced into Taiwan during 1877~1994. It is possible the genes of these exotic pigs introgress into Lanyu pigs after 1877. The aims of the present study were to investigate the genetic variation within conserved Lanyu pigs and the genetic lineage among Lanyu and exotic pig breeds in Taiwan by polymorphisms of microsatellite markers. Genomic DNAs of 242 individuals from Lanyu, Taoyuan, Meishan, Landrace, Duroc, Yorkshire and Berkshire were extracted. Nineteen microsatellite markers were applied in this study that separately located on 15 porcine chromosomes, and were amplified using fluorescence dye labelled primers by PCR. The fragment length polymorphisms of each microsatellites were analyzed by capillary electrophoresis, based on polymorphisms of these microsatellites. The genetic distance were applied for studying the phylogenetic relationships among these pig breeds. The result showed that Lanyu pig had the shortest genetic distance with Meishan (0.2254), Berkshire (0.2351) and Yorkshire (0.2368) compared to the other exotic breeds. The neighbor-joining (NJ) tree was constructed by genetic distance from seven pig breeds with 1000 bootstarp resampling. It showed that the 7 breeds formed 2 major clades (bootstrap value = 99.6), one composed of all the exotic European breeds, and the Asian breeds were classified into another clade, including Lanyu Taoyuan and Meishan pig breeds, and in Asian clade, Lanyu formed a unique subclade (bootstrap value = 73.4). The Neighbor-Joining tree based on genetic distance among 242 individuals of 7 breeds in this study was constructed. All individuals of Lanyu pigs were clustered into a unique clade in NJ tree, suggesting that the conserved Lanyu pigs didn’t suffer gene introgression from exotic pig breeds. Our data showed 15 of 19 microsatellite loci in Lanyu pigs genome deviating from Hardy-Weinberg equilibrium and FIS value of 19 microsatellite loci were all positive, indicated that inbreeding or Wahlund effect during rearing the conserved Lanyu pigs. For the purpose to prevent the inbreeding depression and maintain the genetic diversity of Lanyu pig, the population structure of Lanyu pig was constructed according to microsatellite polymorphisms. The Lanyu pigs was divided as 4 subpopulations, all individuals of NTU herd were formed an independent subpopulation with the probability higher than 80%. The Lanyu pigs in TAPS could be separated into another 3 subpopulations. This result indicated Lanyu pigs in NTU formed the different allele frequency of microsatellite loci from TAPS, the genetic distance between this two populations also supported this result. In conclusion, the Lanyu pig is a unique breed without gene introgression from exotic pig breeds, but it had sustained loss of heterozygosity. In future, these results can be applied in population management of Lanyu pig to maintain the genetic diversity and as a guideline for producing inbreed laboratory Lanyu breed.目錄 頁次 壹、中文摘要 5 貳、英文摘要 7 參、前言 10 肆、文獻檢討 12 1.遺傳分子標記 1.1常用於探討物種間遺傳關係的分子標記 12 1.2 等位基因酶（Allozyme） 12 1.3 DNA序列多型性 13 1.4 DNA重複序列多型性 14 2.蘭嶼豬與其他臺灣地區豬種 2.1臺灣的豬種引進歷史 16 2.2 蘭嶼豬的引進 18 2.3 蘭嶼豬粒線體DNA序列多型性 19 伍、材料與方法 21 1.試驗豬種與來源 21 2.豬隻血液採集 22 3.製備基因組DNA 22 4.微衛星標記引子設計 22 5.聚合酶鏈鎖反應 22 6.毛細管電泳 25 7.軟體分析 25 陸、結果 26 1.蘭嶼豬微衛星基因座多型性 26 2.蘭嶼豬微衛星基因座哈溫平衡測定與FIS值估算 26 3.臺灣本島蘭嶼豬之族群結構估算 26 4.臺東種畜繁殖場與臺大農業試驗場蘭嶼豬兩族群與桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏間遺傳距離 27 5.蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏間遺傳距離估算及建構NJ tree 28 6.蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏於19組微衛星基因座上之FST值 28 7. 建構蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏豬種個體間之NJ tree 28 柒、討論 A..蘭嶼豬族群內微衛星基因座之變異 1.蘭嶼豬保種族群內哈溫平衡檢定與FIS 30 2.蘭嶼豬族群結構與粒線體控制區域序列單倍型 31 3.臺東種畜繁殖場與臺大農業試驗場蘭嶼豬之族群結構與族群間之遺傳關係 32 B. 蘭嶼豬與臺灣外來豬種之遺傳關係 1.蘭嶼豬與約克夏之遺傳關係 32 2. 7個豬種族群間與其個體間根據遺傳距離所建構之NJ tree 33 C. 微衛星多型性應用於族群管理 34 捌、參考文獻 36 玖、表與圖 表1. 試驗之豬隻品種及DNA檢體來源 21 表2. 微衛星標記引子資訊 23 表3. 臺灣本島44頭保種蘭嶼豬19個微衛星基因座上基因多型性 44 表4. 根據19個微衛星標記多型性估算臺東種畜繁殖場蘭嶼豬、臺灣大學農業試驗場蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏族群間遺傳距離 45 表5. 根據19個微衛星標記多型性估算蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏族群間之遺傳距離 46 表6. 蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏族群於19個微衛星標記上之遺傳變異性 47 表7. 臺東種畜繁殖場保種群29頭蘭嶼豬19個微衛星基因座上基因多型性 48 表8. 根據19個微衛星標記多型性估算粒線體控制區域第一型與第二型蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏之遺傳距離 49 圖1. 根據19個微衛星標記多型性估算臺灣本島44頭蘭嶼豬族群結構圖 50 圖2. 利用19個微衛星標記所估算遺傳距離建構蘭嶼豬、桃園豬、梅山豬、藍瑞斯、約克夏、杜洛克與盤克夏之Neighbor-Joining tree 51 圖3. 7豬種間共122個體，根據19個微衛星標記多型性所估算遺傳距離建構之Neighbor-Joining tree 52 圖4. 根據臺灣大學及臺東種畜繁殖場蘭嶼豬個體間之遺傳距離及粒線體控制區域單倍型建構之Neighbor-Joining tree 53725530 bytesapplication/pdfen-US蘭嶼豬微衛星標記多型性哈溫平衡近親配種遺傳距離Lanyu pigmicrosatellite markerpolymorphismsHardy-Weinberg equilibriuminbreedinggenetic distancethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56645/1/ntu-95-R94626016-1.pdf