臺灣博碩士論文加值系統

傳統育種的選種方法在水產養殖上已經行之有年，然而，傳統育種有著選育時間長且需要耗費大量人力等劣勢。故利用分子育種結合傳統育種已成為現今水產養殖選育優良經濟性狀的趨勢。本論文用 11 個龍膽石斑肌肉生長相關基因之多型性微衛星 DNA 標誌分析兩個族群的石斑魚 D79、D132 中各 79 和 95 隻個體的族群遺傳多樣性。我們發現 11 個微衛星標誌在兩個龍膽石斑族群(D79及D132)之平均等位基因數分別為 4.45±2.91與4.36±2.11，平均有效基因數為 2.44±1.39 與 2.66±1.09，平均觀測雜合度為 0.53±0.24 與 0.62±0.27，平均預期雜合度為 0.50±0.19 與 0.55±0.22，平均固定指數為 -0.07±0.25與 -0.09±0.17，平均多樣性訊息含量為 0.50±0.19 與 0.55±0.21。進一步利用 此 11 個肌肉生長相關基因 3’-UTR 之微衛星 DNA 標誌與一個 DNA 缺失 (deletion) 型標誌來分析 D79 及 D132 兩族群龍膽石斑之體重和基因型是否有相關性。結果發現在 D79及 D132 族群各有 9 個及 6 個 DNA 分子標誌在基因型與體重上具有相關性 (p＜0.05)。總結兩個龍膽石斑族群分析結果，發現共有 5 個 DNA 標誌包含 4 個多型性微衛星 DNA (Ela348、Ela9156b、Ela8112a、Ela22361)及 1 個 DNA 缺失標誌 Ela8112d 與其體重性狀有顯著相關，可做為分子標誌以進行龍膽石斑標誌輔助成長育種。其中很有趣的是龍膽石斑 mef2d 3’-UTR 中之多型性微衛星 Ela8112a 及 DNA 缺失標誌 Ela8112d 均可做為標誌輔助育種之第一型分子標誌。進一步利用肌肉專一性表現載體以紅色螢光蛋白做為報導基因，在紅色螢光蛋白 eTcRFP cDNA 後面分別接上龍膽石斑 mef2d 2kb 完整 3’-UTR 及有 51 bp 缺失兩種 3’-UTR 序列，檢視此 mef2d 3’-UTR 之 51 bp DNA 缺失對其在肌肉表現的影響。利用顯微注射將此兩個表現質體分別注入斑馬魚一個細胞期魚卵以檢視其之後在稚魚肌肉的表現，發現轉殖入 eTcRFP cDNA 連接帶有 51 bp DNA 缺失之 mef2d 3’-UTR 的斑馬魚比連接帶有完整 2 kb 3’-UTR 的斑馬魚其紅色螢光表現明顯較弱。結果顯示此 mef2d 3’-UTR 51 bp DNA 缺失可能會經由使 mef2d在龍膽石斑肌肉之表現量變少而影響肌肉生長，且 mef2d 3’-UTR DNA 缺失標誌 Ela8112d 之異型合子即會導致龍膽石斑體重之減少。

Traditional selection and breeding method had been applied in aquaculture for a long time. However, labor and time-consuming are disadvantages for traditional method. Therefore, the combination of molecular breeding and traditional method has become the trend of aquaculture industry to find out valuable traits. In this research, we applied 11 polymorphic microsatellite DNA markers in muscle growth-related genes to analyze 79 and 95 individuals in two populations (D79、D132) of giant grouper (Epinephelus lanceolatus), respectively. Eleven microsatellite markers were used to evaluate genetic diversity between two populations of giant grouper. The results revealed that the mean of observed number of allele, the mean of effective number of allele, the mean of observed heterozygosity, the mean of expected heterozygosity, the mean of fixation index, and the mean of polymorphic information content in D79 and D132 giant grouper populations were 4.45±2.91 and 4.36±2.11, 2.44±1.39 and 2.66±1.09, 0.53±0.24 and 0.62±0.27, 0.50±0.19 and 0.55±0.22, -0.07±0.25 and -0.09±0.17, 0.50±0.19 and 0.55±0.21, respectively. In addition, we applied 11 microsatellite DNA marker and 1 DNA deletion marker in 3’-UTR of muscle growth-related genes of giant grouper to analyze the significance of correlation between genotype and body weight as phenotype. We found out that there are 9 and 6 DNA markers are significantly associated with body weight in the population of D79 and D132, respectively. Five DNA markers including four microsatellite DNA markers Ela348, Ela9156b, Ela8112a and Ela22361 and one DNA deletion marker Ela8112d are significant in both giant grouper populations and can be applied for marker-assisted selection of growth in giant grouper. It’s interesting that the polymorphic microsatellite DNA Ela8112a and DNA deletion marker Ela8112d in the 3’-UTR of giant grouper mef2d could be both used as Type I molecular markers for MAS. To further elucidate the effect of 51 bp DNA deletion in 3’-UTR of mef2d on gene expression in the muscle of fish larva, a muscle-specific expression vector with eTcRFP cDNA as reporter gene linked by 3’-UTR of mef2d with and without 51 bp deletion were injected into one-cell fertilized eggs of zebrafish, respectively. The red fluorescent expression of eTcRFP cDNA linked by mef2d 3’-UTR with 51 bp deletion was much weaker than that linked by intact 2 kb 3’-UTR. The result indicated that the unique 51 bp deletion in mef2d 3’-UTR may reduce its expression in muscle to affect muscle growth of giant grouper and will result in decrease of body weight in heterozygotes of Ela8112d deletion marker.

謝 辭 I
摘 要 II
Abstract III
圖 目 錄 VI
表 目 錄 VII
壹、 前言 1
一、 鞍帶石斑魚簡介 1
二、 傳統育種 1
三、 分子標誌輔助育種 (marker-assisted selection, MAS) 2
四、 分子標誌簡介及演進 3
五、 微衛星DNA (microsatellite DNA) 3
六、 研究目的 4
貳、 材料與方法 5
一、 實驗動物 5
二、 實驗材料 6
1. 實驗質體 6
2. 實驗菌株 6
3. 培養基 6
4. 生物及化學藥品 6
5. 市售生物反應套組 6
6. 儀器設備 7
三、 實驗方法 8
1. 肌肉生長相關基因微衛星標誌分析 8
2. DNA缺失 (deletion) 型標記 (Ela8112d) 分析 12
3. DNA 缺失型標記 (Ela8112d)造成龍膽石斑魚體重差異的初步探討 12
參、 結果 15
一、 肌肉生長相關基因微衛星標誌分析 D79、D132 龍膽石斑魚族群 15
1. 肌肉生長相關基因微衛星標誌在D79、D132龍膽石斑魚族群的遺傳多樣性分析 15
2. 肌肉生長相關基因微衛星標誌在 D79、D132 龍膽石斑魚族群的基因型與性狀 (體重) 相關性分析 16
二、 利用 DNA 缺失型標誌分析 D79、D132 龍膽石斑魚族群 17
三、 DNA 缺失型標記 (Ela8112d) 造成龍膽石斑魚體重差異的初步探討 18
肆、 討論 19
一、 肌肉生長相關基因微衛星標誌分析 D79、D132 龍膽石斑魚族群 19
1. 微衛星標誌於 D79、D132 龍膽石斑魚族群的遺傳多樣性分析 19
2. 微衛星標誌於D79、D132龍膽石斑魚族群基因型與體重相關性分析 21
二、 利用 DNA 缺失型標誌分析 D79、D132 龍膽石斑魚族群 22
三、 DNA缺失 (deletion) 型標記造成龍膽石斑魚體重差異的初步探討 23
伍、 結論 24
陸、 參考文獻 25

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