臺灣博碩士論文加值系統

吳郭魚為全球高經濟性養殖魚種，環境適應力佳、繁殖力強、高產量及高存活率等優點，對於水產養殖選拔育種者來說，成長為一重要之經濟性狀。然而，魚隻成長之體表型變異涉及許多外在環境與內源性荷爾蒙因素之間的相互作用，屬一相當複雜之生物現象。此外，由於雌魚耗費能量於繁殖上，使得成長速度與體型皆較雄魚慢且小。早期利用雌性尼羅吳郭魚與雄性歐利亞吳郭魚之種間雜交可產生近全雄性子代，可保有尼羅吳郭魚成長快速且體型大及歐利亞抗寒的優點
。然而，頻繁的雜交導致漸滲雜交現象，使得單性苗比率已有逐年下降趨勢。因此優質吳郭魚選拔育種改良計畫已是台灣現今刻不容緩的首要任務。透過分子生物學的快速進展，高度多型性遺傳標記已被運用於確認不同吳郭魚品種及雜交家系親屬相關數量性狀之關聯性。利用分子標記輔助種原庫選育之遺傳管理策略，為提升吳郭魚品種改良之重要關鍵，故本研究目的為開發尼羅吳郭魚成長相關之數量性狀基因座標記，利用第I型（prolactin 1-1、prolactin 1-2、gh、igf 1、ghr1、ghr2及progranulin）與第II型（UNH130、UNH146、UNH848、UNH868、UNH898、UNH925、UNH934、UNH1003及GM180）微衛星標記分析尼羅吳郭魚種內雜交（NT1×NT2）之多樣性，於親代發現6個微衛星標記具有差異且於子代族群具有多樣性，可追朔親代傳遞給子代的基因型。遺傳多樣性分析結果，平均觀測雜合度為0.92±0.17，平均預期雜合度為0.60±0.12，平均多態性訊息含量為0.60±0.12，平均固定指數為-0.55±0.21。利用Duncan檢定法對基因型之多重比較結果發現，prolactin 1-2、progranulin及UNH130標記與體重有顯著相關（p<0.01）。進一步於已利用粒線體DNA D-loop PCR-RFLP（HinfI、RsaI、TaqαI）與微衛星標記輔助種別（UNH155、UNH172、UNH773）與性別（ARO114、ARO195、ARO121）鑑定之市售吳郭魚族群中，進行六個月成長試驗分析，相似之試驗結果可獲得驗證。利用GH/IGF-1內分泌系統調節成長相關之gh、igf1、ghr1、ghr2及progranulin等基因標記分析市售吳郭魚不同成長性狀個體之基因表現量結果發現，成長快速組個體腦組織之gh基因表現量高於緩慢組，而於腦與鰓弓組織中皆發現，成長快速組之progranulin基因表現量高於緩慢組。於兩試驗族群之SSR結果發現，prolactin 1-2標記之BB、progranulin標記之BC、UNH130標記之AA、UNH848標記之BC及UNH925標記之CE為成長快速之基因型。本研究開發出可供吳郭魚成長家系選拔育種之數量性狀基因座標記，期望能做為產業評估與追蹤種緣系譜遺傳多型性之指標，除可避免基因窄化所造成之近親衰退外，亦能提高成長性狀改良之篩選效益，未來可提供業者建構完整產銷履歷及品牌認證之科學化管理，為水產養殖產業注入新元素。

Tilapia is the high economically important as food fish species for aquaculture in the world. It has good environmental resilience, strong reproductive capacity, high yield and high survival advantages. Growth trait is an important economic characteristic of selective breeding for aquaculture breeders. However, the phenotypic variation of the normal somatic growth of fish is a remarkably complicated biological phenomenon that involves interactions between external factors and endogenous hormonal factors. In addition, the female fish often consume energy used for breeding, and the growth rates and body size were slower and smaller than males. The use of interspecific hybridization between female Nile tilapia (Oreochromis niloticus) and male Blue tilapia (Oreochromis aureus) are available to product high percentage of male offspring, and retain the advantages of fast-growing, large size, and cold tolerance. However, the interspecific hybridization may lead to the introgression of genes, and the proportion of unisexual seedling has been declining trend. At present, selective breeding for the genetic improvement of quality-based tilapia is an urgent task in Taiwan. Highly polymorphic genetic markers have been used to identify association between markers and quantitative traits related with family relationships in different tilapia species and their hybrids. Genetics and brood-stock management strategies of molecular marker-assisted selection and breeding are important for promoting improved varieties of seeds of tilapia species. The purpose of present study was to develop the quantitative trait locus markers associated with body growth in Nile tilapia. Two strains (NT1 and NT2) of healthy Nile tilapia were bred in conserved stocks at NTOU. The type I (prolactin 1-1, prolactin 1-2, gh, igf1, ghr1, ghr2 and progranulin) and Type II (UNH130, UNH146, UNH848, UNH868, UNH898, UNH925, UNH934, UNH1003, and GM180) microsatellite markers were employed to assess the genotypic polymorphism and to evaluate the growth performance of F1 hybrids from the intraspecies cross family of Nile tilapia. The analysis results of genetic diversity shown that the mean observed heterozygosity was 0.92 ± 0.17, average expected heterozygosity was 0.60 ± 0.12, the average polymorphic information content was 0.60 ± 0.12, and average fixed index was -0.55 ± 0.21. Duncan assay to genotype multiple comparison results shown that, prolactin 1-2, progranulin, and UNH130 loci were significantly associated with body weight (p<0.01). Further subdivided to have used mitochondrial DNA D-loop PCR-RFLP (HinfI, RsaI, TaqαI) and microsatellite markers for species identification (UNH155, UNH172, UNH773) and sexing (ARO114, ARO195, ARO121) in the commercially tilapia population, and the similar results were obtained and verified during the six months of the growing experimental analysis. The GH/IGF-1 endocrine system regulate growth-related genes of gh, igf1, ghr1, ghr2, and progranulin were selected to assess gene expression in different growth trait individuals of commercial tilapia population. The result showed that the gh in brain and progranulin in gill arch were greater expression in fast group than in slow group. Similar results of SSR analysis were found that the BB type of prolactin 1-2, BC type of progranulin, AA type of UNH130, BC type of UNH848, and CE type of UNH925 showed the least suggestive correlations with body weight. The results of the present study suggested that the developed quantitative trait locus markers could be applied to family selection and breeding on the growth traits of tilapia. The technology platform could be developed into an industrial standard for the assessment and evaluation of genetic diversity and to define a marker assisted traceability system for tracing the pedigree to manage the brood-stock resource. It could be not only avoided the inbreeding depression caused by excessive narrowing of the gene pool, but also increased the benefit of selecting for genetic improvement of growth traits. It will provide the industry to build the scientific management of complete traceability and branding certification, and to create an entirely new approach to productivity and competitive advantage in the future.

誌謝 I
中文摘要 II
Abstract III
目錄 V
圖次 VIII
表次 IX
壹、前言 1
一、吳郭魚養殖概況 1
（一）吳郭魚之介紹 1
（二）臺灣吳郭魚養殖 1
（三）單性吳郭魚 2
二、分子育種的發展與應用 3
三、分子標記概述 3
四、標記輔助選育之應用 5
（一）成長性狀鑑定 5
（二）種別鑑定 7
（三）性別鑑定 7
（四）即時定量聚合酶連鎖反應之功能性基因分析 8
五、研究目的 9
貳、材料與方法 10
一、試驗材料 10
（一）生物化學藥品 10
（二）儀器設備 10
（三）試驗生物 10
（四）試驗環境 11
二、試驗流程 12
三、試驗分析引子 13
四、試驗方法 16
（一）基因組DNA萃取 16
（二）引子設計 16
（三）標記基因型判讀與分析 17
（四）種別鑑定與性別分析 18
（五）功能性基因表現之即時定量聚合酶連鎖反應分析 19
參、結果 21
一、尼羅吳郭魚種內雜交（NT1×NT2）家系親代與子代之遺傳多樣性分析
21
二、尼羅吳郭魚種內雜交（NT1×NT2）家系子代之成長性狀分析 21
三、尼羅吳郭魚種內雜交（NT1×NT2）家系子代之成長相關基因型分析 22
（一）第一型微衛星標記與體重相關性分析 22
（二）第二型微衛星標記與體重相關性分析 22
（三）微衛星標記組合與成長性狀相關性分析 23
四、市售吳郭魚族群之成長試驗分析 23
五、市售吳郭魚族群之種別與性別分析 23
（一）粒線體DNA D-loop PCR-RFLP分析結果 23
（二）微衛星標記之鑑種與性別分析結果 24
六、市售吳郭魚族群之功能性基因分析 24
七、市售吳郭魚族群之成長相關基因型分析 24
（一）市售吳郭魚族群之遺傳多樣性分析 24
（二）市售吳郭魚族群之成長相關基因型分析 25
八、尼羅吳郭魚種內雜交（NT1×NT2）與市售吳郭魚族群成長相關基因型
之比較
26
肆、討論 27
一、尼羅吳郭魚與市售吳郭魚族群之遺傳多樣性 27
二、DNA遺傳標記輔助吳郭魚族群種緣與性別鑑定 28
三、吳郭魚功能性基因表現之基因型與成長性狀相關性 28
伍、結論 31
陸、參考文獻 32
附錄 68

 

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