| 中文摘要 |
台灣鯛為農委會當前積極推廣之外銷四大旗艦產品之一。然而近來兩岸走私猖獗，使得銷往國外之冷凍台灣鯛多次被扣關，而被扣關的貨品中，絕大部分非台灣所養殖。雖然台灣吳郭魚繁養殖技術早已受到肯定，目前仍無基因品系追蹤觀念來強化養殖水產品的管理保護與來源追朔。微衛星與單一核甘酸多型性基因標記是目前被認為可用於基因品系追蹤與食品來源追朔，也是生物技術在傳統育種過程的最佳輔助工具，除可做為物種親緣關係鑑定、避免近親交配，更可提早從育種的子代中選取所需的重要遺傳性狀。由於微衛星本身具有物種專一性及種間差異性、種類繁多、高度多型性…等特性，因此初期收集不易。本計畫共分成兩大部份，第一部份利用基因序列與微衛星基因座區分不同品種吳郭魚，利用聚合?鏈反應增幅出約280bp的基因體DXTU1片段，發現核?酸多型性可區分四種吳郭魚品種與台灣鯛；第二部份，利用台灣鯛全基因體磁性雜交移除複製技術，經由去氧核醣核酸定序及比對分析結果，目前已得到富含二核?(ag, ga, ct, ac, ca, tc, gt, at)、三核?(atc, gat, tta, tga, cat, tca, atg, gag,)、四核?(ccat, atcc, ggat, catc, tcca, ctga, gatg, )，以及少數寡核?序列aaaac, atcatc, acaaaa, caaaaac, tctgtctccttgt, tccatccgttga, ataataataata, gaagataagactta, tttcacttttatcaatt, gttttgtgttgggtgtgtttgg…等的重覆序列以及他們的外圍DNA序列。利用本計畫所獲得之四個微衛星DNA基因座片段，設計引子進行聚合?鏈反應，可鑑別不同品種之吳郭魚與台灣鯛。初步序列分析將可用於逐步朝向建立臺灣鯛專一性去氧核醣核酸指紋序列和已知品系的吳郭魚做品系確認比對。 |
| 英文摘要 |
Taiwan tilapia is one of the four major export products actively promoted by Council of Agriculture. However, frozen tilapia have been detained by Customs of EU countries due to the illegal smuggling from the Mainland China. Although tilapia is well cultured in Taiwan, but the genetic tracing technology is seldom applied in this species. The tool can be highly complementary to all traditional breeding programs based upon phenotypic strategies by providing suitable or good polymorphic loci to track stock parentage relationship and minimizing effects of inbreeding. Two types of DNA markers have been suggested in traceability schemes, microsatellites and single nucleotide polymorphisms (SNPs). This project include two parts, first part use the sequence variations and microsatellite polymorphism to distinguish tilapia species. A 280bp fragment of the DXTU1 locus was amplified by the polymerase chain reaction from different tilapia species, and different nucleotide polymorphism marker could be defined four different tilapia species. Second part, the whole genome amplification technique was applied in Taiwan tilapia to extract microsatellite DNA by magnetic hybridization subtraction of conserved biotinylated degenerated oligo. DNA cloning and sequencing revealed highly enriched in microsatellite DNA repeat sequence of dinucleotide (ag, ga, ct, ac, ca, tc, gt, at), trinucleotide (atc, gat, tta, tga, cat, tca, atg, gag, ), tetranucleotide (ccat, atcc, ggat, catc, tcca, ctga, gatg, ), oligos (aaaac, atcatc, acaaaa, caaaaac, tctgtctccttgt, tccatccgttga, ataataataata, gaagataagactta, tttcacttttatcaatt, gttttgtgttgggtgtgtttgg…etc), and their flanking sequence. Four microsatellite DNA sequences obtained in this project can be used to discriminate tilapia species by their different polymorphic loci. The methodology developed here was highly efficient and can be applied in genetic tracing and breeding program of Taiwan tilapia. |