臺灣博碩士論文加值系統

銀紋笛鯛 (Lutjanus argentimaculatus) 廣泛分布於熱帶與副熱帶的印度-西太平洋區，是我國西南沿海重要且常見的高經濟價值的漁獲物種之一。臺灣身處亞熱帶地區，四面環海，周邊漁業資源多樣性豐富，近年來隨著人民生活水準提高，加速漁業技術的發展進度，沿近海漁業資源在長期過度捕撈下，資源量明顯萎縮，因此藉由大量放流人工培育種苗來增殖漁業資源是最有效、最直接的方法之一，然增殖放流前應先瞭解其野生族群結構及漁業資源等生物學相關研究以利後續評估。本研究以銀紋笛鯛 (Lutjanus argentimaculatus) 為目標物種，利用耳石判讀齡後所得之成長參數及微衛星基因座基因型差異，來解析台灣周邊海域銀紋笛鯛之野生個體族群結構，進一步了瞭解各地區間是否有族群遺傳結構的差異，以提供往後增殖放流時訂定規範之參考依據。
本研究於2015年3月至2016年7月間分別於台灣的周邊海域進行採樣，將台灣劃分成五個區域，分別為東北、北、中、南部及澎湖，共採集405尾野生樣本，每尾皆測量其外部型態，採集魚鰭組織及耳石等生物學樣本，利用其中278顆耳石切片並計算邊緣成長率 (MIR)，結果顯示其耳石暗帶為一年形成一輪，且於4至7月間形成。而後利用耳石做為定齡之應用，在五個地區中年齡頻度結果顯示1至7歲的樣本佔將近全體95%，其中2歲之樣本最多，佔全部的39%。以變積分析 (ANCOVA) 檢定體長體重關係式，結果顯示只有東北部及南部地區間的成長率有顯著差異 (F1,188= 6.503, p < 0.05)，而在Phi primer test的計算數值上，其地區間的φ’值相差介於 0.0至0.4，故就本研究採集的樣本中所分析的成長數據來看，台灣周邊海域野生銀紋笛鯛在成長上有地區間的差異，但是否已分化為不同系群或種群，需要進一步的探討與研究。
本研究進一步利用次世代測序技術 (NGS, Next Generation Sequencing) 開發了16組微衛星基因座 (microsatellite loci) 引子，在其中4組微衛星的基因型分析結果顯示，北部和南部及澎湖地區的野生族群並無顯著差異，唯獨和中部地區於2016年雲林所採集的樣本有顯著的遺傳差異 (p < 0.001)。其中養殖場的種魚和台灣周邊海域的野生族群有顯著差異(p < 0.005)，但與中部地區的遺傳結構差異較小。綜合本研究以年齡成長與次世代測序技術的結果為依據，將耳石所分析的成長參數及微衛星基因型分析應用在解析銀紋笛鯛野生族群結構是可行的。

The mangrove red snapper (Lutjanus argentimaculatus) is widely distributed in tropical and sub-tropical Indo-West Pacific. It is an econonmic and valuable species in Taiwan. Taiwan is located in subtropical regions and surrounded by the ocean, the fisheries resource is highly diversed. In recent years, the industry developed and development of fishery technology casued fishery resource decreased. Releasing hatchery fish fry is one of the most effective methods for sea ranching. However, the studies of wild population structure need long-term evaluation. In this study, growth parameter and genetical analytical methods were applied to analyze mangrove red snapper (Lutjanus argentimaculatus) population structure around Taiwan.
A total of 405 specimens were collected from northeast, northern, central, southern Taiwan and Penghu between March 2015 and July 2016. Total length of each individuals were measured, and biological tissue and sagittal otolith were collected. Age of 278 fish was read from otolith sections. Otolith opaque zones appeared on the edge from April to July, and the annulus forms one ring per year. The results showed that the age structure of the 2 - year - old samples were the most abundant, accounting for 39%, of which 1 - 7 years old accounted for nearly 95%. The results showed a significant difference between the northeastern and southern regions (F1,188= 6.503, p < 0.05), and in the Phi primer test calculation, the values is range from 0.0 to 0.4 in the five range. Based on the data collected from the samples collected in this study, there may be differences between individuals in the wild population of mangrove red snapper around Taiwan, but whether it has been diversed into populations need further investigate.
We have also developed 16 microsatellite loci primers by using NGS for further population structure investigation. The genotyping results from 4 microsatellite loci showed that there was no significant difference among the wild populations, but only the central region (Yunlin) in 2016 (p < 0.001). However, aquaculture populations are significant different to wild populations (all p < 0.005). The results suggest that the otolith growth parameters and microsatellite genotypes are feasible in the analysis of the wild population structure of mangrove red snapper.

摘要 I
Abstract II
壹、 緒論 1
一、 銀紋笛鯛 1
1.1.1生物學及地理分布 1
1.1.2生活史模式 1
1.1.3經濟性利用到科學研究 2
二、 耳石 3
1.2.1型態及功能 3
1.2.2耳石結構 3
1.2.3年齡形質應用 4
三、 微衛星DNA 5
1.3.1 簡單重複序列 5
1.3.2 中性分子遺傳標誌 5
四、 研究動機及目的 7
貳、 研究方法 8
一、 樣本採集 8
2.1.1採樣時間地點 8
2.1.2魚體樣本處理 8
二、 耳石量測 8
2.2.1耳石製備 8
2.2.2年輪驗證 9
2.2.3年輪及輪寬讀取 9
2.2.4體長回推及成長方程式參數之推估 10
2.2.5成長參數之驗證 10
三、 基因檢測 10
2.3.1 DNA 萃取 10
2.3.2以次世代測序開發微衛星基因座 11
2.3.3聚合酶連鎖反應 (Polymerase chain reaction, PCR ) 11
2.3.4螢光毛細電泳分析 12
四、 資料分析 12
2.4.1 體長-體重關係式 12
2.4.2體長-耳石徑關係式 12
五、 統計分析 12
參、 研究結果 13
一、 樣本組成 13
二、 體長-體重關係 13
三、 邊緣成長率 (MIR) 14
四、 年齡頻度 14
五、 成長方程式 15
六、 成長參數驗證 16
七、 微衛星標記引子對設計 16
八、 族群遺傳結構分析 17
肆、 討論 17
一、 耳石處理及定齡 17
二、 年齡成長方程式 18
三、 成長係數驗證 19
四、 野生族群遺傳結構 19
五、 種間遺傳差異 19
伍、 結論 21
參考文獻 22

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