林恩仲Lin, Ec-Chung臺灣大學:動物科學技術學研究所呂岳錚Lyu, Yue-JhengYue-JhengLyu2010-05-112018-06-292010-05-112018-06-292009U0001-2407200921073900http://ntur.lib.ntu.edu.tw//handle/246246/182088近年來由於狂牛病的爆發及各種食品安全事件，使得食品安全日漸受到重視，故最近世界各國以歐盟為首，都在推行產銷履歷制度以增進食品安全。傳統使用在動物製品上的產銷履歷以紙本記錄及耳標為主，但是此兩種方法均有造假或錯置的可能，因此利用 DNA 分子標幟進行遺傳追溯對傳統產銷履歷方法進行驗證成為近年來的研究方向之一，由於微衛星標幟具有高多態性、廣泛分布於真核生物染色體及易於使用聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 技術複製等優點，使之常被用來做為遺傳追溯的分子標幟，本研究之目的即在建立可用於台灣地區豬隻產銷履歷驗證之微衛星標幟組。 本研究對來自檢定站，台灣地區常見的三個品種的純種豬隻 (藍瑞斯, 約克夏及杜洛克) 以及來自數個私人豬場之商用雜交肉豬 (藍瑞斯×杜洛克 (LD)，藍瑞斯×約克夏×杜洛克 (LYD) 及未知遺傳背景雜交肉豬 (UnP) 採用10 組微衛星標幟進行研究，計算各標幟之個體鑑別率 (probability of identity, P(ID)) 以確定其個體鑑別之效率，實驗方法為對微衛星標幟引子標記螢光進行兩組多引子PCR 增殖，並進行毛細管電泳以進行基因型鑑定，結果發現三個純品種其理論異質度 (expected heterozyosity, HE) 在無遺傳相關個體上分別為 0.62、0.65 及 0.65；在所有個體間 (部分具有遺傳相關) 分別為 0.61、0.66 及 0.65。其多態性訊息含量 (polymorphism information content, PIC) 在無遺傳相關個體上分別為0.57、0.60 及 0.60；在所有個體間 (部分具有遺傳相關) 分別為 0.57、0.61 及 0.61，三個純品種均在 S0227 有最低的遺傳岐異而在 SW857 有最高的遺傳岐異，進行哈溫平衡 (Hardy-Weinberg equilibrium, HWE) 檢定的結果，在無遺傳相關個體上，藍瑞斯及約克夏各有一個標幟，杜洛克有三個標幟偏離 HWE (P<0.05)，在所有個體間 (部分具有遺傳相關)，藍瑞斯有一個標幟，約克夏及杜洛克各有三個標幟偏離 HWE (P<0.05)，但無論有無遺傳相關，十個標幟之總合均無族群偏離 HWE，表示少數標幟偏離 HWE 對個體鑑別之影響並不顯著。 進行個體鑑定之結果，三個品種其 P(ID) 在遺傳相關個體上分別為 1.42×10-8、6.49×10-9 及 1.23×10-8，在所有個體間 (部分具有遺傳相關) 分別為 1.58×10-8、5.61×10-9 及 9.34×10-9 ，而對雜交豬隻進行個體鑑別之結果，LD 雜交肉豬、LYD 雜交肉豬及 UnP 雜交肉豬其 P(ID) 分別為1.79×10-9、6.14×10-9 及 3.83×10-9，所有族群之 P(ID) 值均小於1×10-7。於屠宰場採集民間兩豬場 14 批未知品種肉豬，共 339 頭，與大賣場取得之零售樣品共 8 批進行產銷履歷驗證，可以發現大賣場採得之同批樣品內均有相同之基因型，但僅有一批大賣場樣品可追溯到正確批次之屠宰場採集樣品。其餘之大賣場採樣樣品均未能對應到正確之屠宰場採樣樣品，這些樣品有些具有銷售日期與屠宰日期相差過遠之現象，或是經基因型鑑定的結果，出現了屠宰場採樣樣品所來自之兩豬場所無或極少出現之交替基因。 上所述，臺灣近五年之每年上市屠宰的純種淘汰猪及雜交肉猪總數在一千萬頭以下，故屠宰猪在無論有無遺傳相關的情況下，此 10 組微衛星標幟應可達成臺灣常見之純種猪及雜交肉猪的個體鑑別需求，以應用於未來新鮮猪肉產品的產銷履歷回溯驗證。In the last few decades, Bovine spongiform encephalopathy (BSE) and other food safety issues induced to increase consumer’s concerns of safety in food supply chain. EU member countries and other nations in the world have regulations to ensure food traceability in order to promote food safety. Conventional traceability of animal products consists of labeling system and paper documents, which could be counterfeit and less reliable. To verify the correctness of conventional traceability, genetic traceability based on DNA markers has become popular in recently year. Because microsatellite markers have advantage such as highly polymorphic, abundantly dispersed throughout most eukaryotic nuclear genome, and efficiently amplified using polymerase chain reaction (PCR), they are often utilized as molecular markers in genetic traceability. The objective of this study is to establish the microsatellite marker sets for genetic verification traceability of fresh pork products in Taiwan.n this study, purebred pigs of three breeds (Landrace, Yorkshire, and Duroc) from the central test station and commercial crossbred pigs (Landrace × Duroc (LD), Landrace × Yorkshire × Duroc (LYD), and unknown genetic background crossbred pigs (UnP) from several private farms were genotyped using ten microsatellite marker sets. The probability of identity (P(ID)) of each marker set in individual genetic traceability was calculated. We performed two multiplex polymerase chain reaction (PCR) with fluorescent-labeled microsatellite markers. The PCR products were then separated by capillary electrophoresis and genotyping. The expected heterozygosity (HE) in none genetic related individuals of three pure breeds were 0.62, 0.65, and 0.65; in all individuals (partial genetic related) of three pure breeds were 0.61, 0.66, and 0.65. The polymorphism information content (PIC) in unrelated individuals of the three breeds were 0.57, 0.60, and 0.60; in all individuals (partial genetic related) of three breeds were 0.57, 0.61, and 0.61. In the three breeds, the genetic diversity was the lowest in the marker of S0227 and the highest in SW857. After performing Hardy-Weinberg equilibrium (HWE) test in the three breeds, in unrelated individuals of the three breeds, there were one in Landrace, one in Yorkshire, and three in Duroc microsatellite markers against HWE respectively (P<0.05), in all individuals (partial genetic related) of three breeds, there were one in Landrace, three in Yorkshire and three in Duroc microsatellite markers against HWE respectively (P<0.05). The combination of observed heterozygosity (HO) with ten microsatellite marker sets showed that the overall situation still agreed with HWE. Therefore, those marker sets against HWE had no significant effects on individual identification. he results of individual identification showed that P(ID) in unrelated individuals of three breeds were 1.42×10-8, 6.49×10-9, and 1.23×10-8; in all individuals (partial genetic related) of three breeds were 1.58×10-8, 5.61×10-9, and 9.34×10-9. The P(ID) of LD crossbred pigs, LYD crossbred pigs, and UnP were 1.79×10-9, 6.14×10-9, and 3.83×10-9, respectively. All the tested groups had their P(ID) values less than 1.00×10-7. We collected 339 UnP with 14 batches from two different private farms in slaughter house, and 8 batches of retail sale sample from hypermarkets for traceability test. The result showed that samples from hypermarkets with the same batch number had the same genotypes. Two batches from hypermarkets could trace back to batches from slaughter house correctly. Other samples from hypermarket could not correspond to correct sample collected in slaughter house, some of the samples had issues of the sales date far from slaughtering date, the results of genotyping, the alleles appeared in these samples were rare or even never appeared in these two farms.n Taiwan, the number of purebred and crossbred pigs slaughtered per year has been less than ten million (1×107) heads in recent five years. Thus, according the results of this study these ten microsatellite markers are sufficient for individual identification among the common purebred and crossbred commercial pigs in the genetic traceability of fresh pork.誌謝....................................................................................................................................Ι錄..................................................................................................................................II次..................................................................................................................................V次.................................................................................................................................VI 文摘要......................................................................................................................VIII文摘要..........................................................................................................................X、前言...........................................................................................................................1、文獻檢討.....................................................................................................................2、產銷履歷 (Traceability) 的重要性.......................................................................2、產銷履歷之原則及精神.........................................................................................3、各國產銷履歷之執行現況.....................................................................................4一) 歐洲地區..........................................................................................................4二) 北美地區..........................................................................................................5. 美國..................................................................................................................5. 加拿大..............................................................................................................8三) 亞洲地區..........................................................................................................8 1. 日本..................................................................................................................8 2. 台灣..................................................................................................................9四) 其他地區........................................................................................................14、分子遺傳標記.......................................................................................................14一) 等位基因酶 (Allozyme)................................................................................15二) DNA 分子遺傳標記.......................................................................................15. 限制片段長度多態性 (Restriction Fragment Length Polymorphism, RFLP).............................................................................................................................16. 隨機增殖 DNA 多態性 (Random Amplified Polymorphism DNA, RAPD).............................................................................................................................16. 增殖片段長度多態性 (Amplified Fragment Length Polymorphism, AFLP)..............................................................................................................................17. 微衛星 (Microsatellite).................................................................................18. 單核苷酸多態性 (Single Nucleotide Polymorphism, SNP).........................19(三) 族群遺傳統計..............................................................................................20. 有效交替基因數 (Number of effective alleles, Ne)......................................20. 理論異質度 (Expected heterozygosity, HE)..................................................21. 觀測異質度 (Observed heterozygosity, HO)............................................... 21. 多態性訊息含量 (Polymorphism Information Content, PIC)......................22. 懷特氏固定指數 (Wright’s fixation index)..................................................22、分子遺傳標記於產銷履歷上之運用...................................................................23、研究動機...............................................................................................................26、材料與方法...............................................................................................................28、試驗樣本..............................................................................................................28、基因組 DNA 萃取...............................................................................................28、微衛星標幟引子設計...........................................................................................30、聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR).........................................31、電泳樣品製備.......................................................................................................33、PCR產物基因型判別...........................................................................................33、微衛星標幟基因型頻率統計...............................................................................33、結果...........................................................................................................................35、PCR產物增殖結果...............................................................................................35、純種豬隻之遺傳多態性 .....................................................................................35、純種豬隻之 HWE 檢定及 FIS 值......................................................................44、純種豬隻之個體鑑定...........................................................................................47、雜交豬隻之個體鑑別...........................................................................................50、產銷履歷之驗證...................................................................................................53、討論...........................................................................................................................56、純種豬隻微衛星標幟多態性之討論...................................................................56、純種豬隻之 HWE 檢定......................................................................................57、純種豬隻之個體鑑別...........................................................................................58、雜交豬隻之個體鑑別 .........................................................................................60、驗證結果之探討...................................................................................................61、未來研究方向.............................................................................................62、結論...........................................................................................................................63、參考文獻...................................................................................................................64次次 1. 產銷履歷流程圖....................................................................................................4 2. 產銷履歷農產品 TAP, OTAP及UTAP 標章 ..................................................10 3. 各微衛星標幟其PCR產物所標定之螢光及其片段大小..................................34 4. 毛細管電泳訊號經 Peak Scanner 1.0 分析之波型圖.......................................39圖 5. 純品種豬隻 P(ID) 對標幟數之折線圖...............................................................52 6. 純品種及雜交豬隻 P(ID) 對標幟數之折線圖....................................................52次次 1. 歐盟近年推行之食品安全措施一覽表................................................................7 2. 美國近年推行之食品安全措施一覽表................................................................7 3. 日本近年推行之產銷履歷措施一覽表................................................................9 4. 本實驗所使用豬隻品種及 DNA 樣品來源......................................................29 5. 屠宰場採樣之日期、批號及樣品數....................................................................30 6. 大賣場採樣之地點、批號、產品類型及採樣號..................................................31 7. 本試驗 PCR 反應所使用之微衛星標幟、螢光種類及其引子序列.................32 8. 本試驗所觀測到各微衛星標幟之交替基因 (PCR 產物片段大小)................38 9. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體在 10 組微衛星標幟之Na.....................................................................................................................40 10. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之全部之個體在 10 組微衛星標幟之Na...............................................................................................................40 11. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體在 10 組微衛星標幟之 Ne.............................................................................................................41 12. 藍瑞斯、約克夏及杜洛克三個品種豬隻之全部之個體在 10 組微衛星標幟之 Ne.................................................................................................................41 13. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體在 10 組微衛星標幟之 HE 及其標準機差 (SE).........................................................................42 14. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之全部之個體在 10 組微衛星標幟之 HE 及其標準機差 (SE)..........................................................................42 15. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體在 10 組微衛星標幟之 PIC...........................................................................................................43 16. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之全部之個體在 10 組微衛星標幟之 PIC...........................................................................................................43 17. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體 10 個微衛星標幟之 HO................................................................................................................45 18. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之全部之個體 10 個微衛星標幟之 HO................................................................................................................45 19. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體 10 個微衛星標幟之 FIS.................................................................................................................46 20. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之全部之個體 10 個微衛星標幟之 FIS.................................................................................................................46 21. 藍瑞斯、約克夏及杜洛克三個純品種豬隻之無相關個體 10 個微衛星標幟之 P(ID)..............................................................................................................48 22. 藍瑞斯、約克夏及杜洛克三個純品種豬隻全部之個體 10 個微衛星標幟之 P(ID)....................................................................................................................48 23. 藍瑞斯、約克夏及杜洛克三個純品種豬隻無相關個體 10 個微衛星標幟之 P(ID)sibs................................................................................................................49 24. 藍瑞斯、約克夏及杜洛克三個純品種豬隻全部之個體 10 個微衛星標幟之 P(ID)sibs................................................................................................................49 25. 各品種以及雜交豬隻之各項統計....................................................................51 26. 本試驗A及B兩豬場樣品微衛星標幟交替基因頻率.....................................53 27. 大賣場採樣之基因型鑑定結果........................................................................55 28. 產銷履歷之驗證結果........................................................................................55 29. 單一族群及其內部兩個次族群交替基因頻率及 HE......................................58application/pdf555089 bytesapplication/pdfen-US猪微衛星標幟產銷履歷個體鑑別PigsMicrosatellite markersTraceabilityIndividual identification[SDGs]SDG2thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182088/1/ntu-98-R95626009-1.pdf