臺灣博碩士論文加值系統

本研究探討台灣鮭魚保育上的3個議題：(1)櫻鮭家族成員間之親緣關系；(2)遺傳多樣性的調查；(3)台灣鮭魚性別比例的推估。
使用AFLP分子標記分析四個櫻鮭家族（Oncorhynchus masou complex）成員的親緣關係，並以Nei遺傳距離（Nei's genetic distance）與鄰接法（Neighbor-joining method）所建構出之演化樹（Phylogenetic tree）中，台灣鮭魚（O. m. formosanus）為最早分化出之亞種，其次為櫻鮭（O. m. masou），最後為琵琶鮭（O. m. subsp.）和石川鮭（O. m. ishikawae）。此結果不但顯示台灣鮭魚並非櫻鮭之地域族群（Local population），同時也排除台灣鮭魚是近代引進之外來物種的可能性。據此結果推測，台灣鮭魚應源於櫻鮭家族之共同祖先，而非源於櫻鮭。由於台灣鮭魚自然陸封於台灣大甲溪上游並且獨立演化，因此需視為顯著演化單元（Evolutionarily Significant Unit）採取保育措施。此外，本研究也發現數條台灣鮭魚的AFLP特有條帶，可輔助傳統分類方法之不足，有效鑑別櫻鮭家族的成員。
在台灣鮭魚遺傳多樣性的調查中，於2004年及2005年分別自七家灣溪1至3號壩間採集了30尾及28尾台灣鮭魚樣本。以微衛星標記（Microsatellite）和AFLP進行分析，27個微衛星位點中僅有1個微衛星位點具有2個等位基因，展現多樣性，其餘位點皆為同質，平均基因多樣性指數（He）分別為0.0071與0.0067；使用3個AFLP選擇性引子對，共得到196條擴增片段，多樣性片段為58條(29.59%)，基因多樣性指數為0.045及0.054。微衛星及AFLP數據皆顯示台灣鮭魚遺傳變異極低。比較二年的AFLP數據可以發現，雖然基因多樣性指數並未下降，但2004年樣本具有的多樣性位點約有3分之1在2005年樣本中消失或固定，另外3分之1的位點頻率大幅改變，呈現隨機飄變的現象。此結果顯示台灣鮭魚遺傳多樣性正持續喪失，個體趨於同質。
本研究開發出以PCR鑑定台灣鮭魚性別的方法。此方法具有不受季節、發育時間等限制的優點，其準確性高達98.32%（124個樣本中有二個樣本的表型與基因型不符）。2004至2008年的野外台灣鮭魚族群的性比（雄/雌）分別為0.59、0.50、1.19、0.57、0.72，其中2007年的性比顯著偏離1：1 (P < 0.05)。此結果顯示台灣鮭魚的性比並非固定於1：1，在有效族群的計算上，應考量到性比不均的情況。
總結來說，台灣鮭魚為櫻鮭家族中的顯著演化單元，在遺傳及地理分布上均有其特殊性，在保育措施（例如：野外族群監測、人工繁殖放流、精液凍結保存等）實行的同時，AFLP及性別標記可有效進行遺傳管理，保存最大基因多樣性，希望達到保全台灣鮭魚永續生存的目標。

This study aimed at investigating three conservation issues on Formosa landlocked salmon: (i) the phylogenetic relationship between Formosa landlocked salmon and other subspecies of Masu salmon complex (Oncorhynchus masou complex), (ii) the genetic diversity that exists in Formosa landlocked salmon, and (iii) the estimation of the sex ratio of Formosa landlocked salmon.
The phylogenetic tree is reconstructed based on AFLP data and by using neighbor-joining method and Nei's genetic distance. In this tree, Biwa salmon (O. m. subsp.) and Amago salmon (O. m. ishikawae) are clustered first, Masu salmon (O. m. masou) are clustered second, and finally Formosa landlocked salmon (O. m. formosanus) are jointed to this cluster. The high bootstrap values at each node indicate that this tree is robust. The results show that instead of dividing from Msau salmon, Formosa landlocked salmon is the first subspecies divided from ancestor of Masu salmon complex. Formosa landlocked salmon is considered as an evolutionarily significant unit (ESU) due to geographic separation and genetic differentiation among Masu salmon complex. Furthermore, several subspecies specific bands, which are helpful for distinguishing subspecies from each other, are found in each member of Masu salmon complex.
Genetic diversity of Formosa landlocked salmon in Chichiawan Stream are examined by using AFLP and Microsatellite markers. For consecutive two years (2004 and 2005), a total of fifty-eight individuals (thirty in 2004 and twenty-eight in 2005) were examined. Of twenty-seven Microsatellite loci, only one locus was polymorphic with two alleles while others were monomorphic. The average He are 0.0071 and 0.0067, respectively. One hundred and ninety six loci were detected using three AFLP selective primer pairs, of which fifty-eight were polymorphic with a proportion of 29.59%. The average He are 0.0045 and 0.054, respectively. Both Microsatellite and AFLP data show the low genetic diversity in Formosa landlocked salmon. Comparison of allele frequency of fifty-eight AFLP polymorphic loci between two years, eighteen (about one third) of the amplified polymorphic loci became fixed (p = 100%) or lost (p = 0%) and another sixteen (about one third) was changed in the 2005 population. The results indicate that Formosa landlocked salmon are losing genetic diversity due to genetic drift.
In this study, a simple, reliable, non-invasive PCR method is developed to identify sex of Formosa landlocked salmon. The reliability of sex identification is 98.40% (1.60% mismatch; two mismatches in 125 individuals) for Formosa landlocked salmon. The sex ratios (male/female) of field Formosa landlocked salmon from 2004 to 2008 were 0.59, 0.50, 1.19, 0.57 and 0.72, respectively. The sex ratio of 2007 was significant differed from 1:1 (p<0.05). The results indicate the sex ratio of Formosa landlocked salmon is not consistent with 1:1. Biased sex ratio has to be considered when effective population size is counted.
In summary, Formosa landlocked salmon is an ESU of Masu salmon complex and should be conserved. In conservation program, AFLP and sex marker are useful tools for genetic management and conserving maximum genetic diversity.

謝辭.......................................................i
中文摘要...................................................ii
英文摘要..................................................iii
目錄......................................................iv
表目錄.....................................................v
圖目錄....................................................vi
第一章 前言.................................................1
第一節 台灣鮭魚簡介........................................1
第二節 台灣鮭魚的生存危機...................................3
第三節 生物多樣性與保育遺傳學...............................6
第四節 分子標記簡介........................................9
第五節 性別鑑定在保育生物學的重要性.........................11
第六節 太平洋鮭魚的性別決定機制及性別標記....................12
第七節 研究目的..........................................13
第二章 材料方法............................................15
第一節 樣本來源及採集地點..................................15
第二節 粹取樣本基因組DNA..................................16
第三節 AFLP指紋圖譜建構...................................17
第四節 AFLP數據分析......................................19
第五節 微衛星DNA圖譜建構..................................22
第六節 開發台灣鮭魚的性別標記..............................22
第三章 結果................................................25
第一節 櫻鮭家族的親緣關係..................................25
第二節 台灣鮭魚的遺傳多樣性................................26
第三節 台灣鮭魚性別標記與性比..............................27
第四章 討論................................................30
第一節 櫻鮭家族的親緣關係..................................30
第二節 台灣鮭魚的遺傳多樣性................................32
第三節 台灣鮭魚性別標記與性比..............................35
第四節 台灣鮭魚的未來.....................................38
第五章 結論................................................40
參考文獻...................................................41
圖........................................................50
表........................................................67
附錄1台灣鮭魚的分子遺傳標記－預備實驗.........................80
附錄2黑鯛精液樣本凍結前後的AFLP測試..........................83
附錄3 Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation...............85
作者簡歷...................................................90

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