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研究生:戴澤宇
研究生(外文):Tse-Yu Tai
論文名稱:以龍膽石斑轉錄體發展標誌輔助育種之生長相關第一型微衛星DNA標誌
論文名稱(外文):Development of Growth-Related Type I Microsatellite DNA Markers from the Transcriptome of Giant Grouper for Marker-Assisted Selection
指導教授:龔紘毅
指導教授(外文):Hong-Yi Gong
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:100
中文關鍵詞:分子標誌微衛星序列龍膽石斑轉錄體分子標誌輔助育種
外文關鍵詞:molecular markermicrosatellitesGaint Grouper TranscriptomeMAS
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本研究主要以龍膽石斑轉錄體49,722個有註解Unigenes,共兩萬多非重複龍膽石斑基因並以GeneOntology (GO) 分析歸類,建立微衛星序列資料庫作為平台輔助傳統育種,共4701個微衛星DNA。其中di- , tri- , tetra- , penta- , hexa-nucleotide repeat SSRs分別有1779、1996、247、113與566個。2729個可設計primers進一步研究,其中微衛星序列位於5’ -UTR為495個,位於3’ -UTR 862個,位於蛋白質轉譯區域有1218個。將2729 cDNA以GO分類,其中與骨骼肌生成控制相關基因的微衛星有73個,加上其他與生長也有關係的基因上9個SSR,經過試驗可以成功擴增的55個SSR有31個具有多型性 (polymorphisms),其觀測雜合度 (Ho) 0.01-0.7與期望雜合度 (He) 0.01-1.0,結果顯示位於 3’-UTR 的 SSR 較容易具有多型性。我們成功從一批傳統育種法選出成長快與慢的魚苗中發展10個多型性、基因型與體重有顯著關係的分子標誌 (P<0.05) Ela8112d與9個 SSR (Ela8112a, Ela9156b, Ela28217, Ela22373, Ela28216, Ela25872, Ela348, Ela13962, Ela22361)。Ela8112d是我們意外發現在其3’ -UTR (GT)n SSR序列旁具有一51鹼基對缺失。在生長較慢的石斑魚有此缺失的異型合子基因型 (heterozygotes) 頻率偏高,而成長較快的魚有缺失的異型合子基因型頻率偏低。並繼續分析另一批以傳統法挑選成長快速的魚苗。顯示這傳統選育的成長快速族群內符合5個微衛星分子標誌 (Ela9156b、Ela28216、Ela348、Ela13962、Ela22361)與1個缺失基因分子標誌Ela8112d,初步證實分子標誌與此育種平台的可用性,未來應可發展其他經濟性狀相關的分子標誌,選育各種不同的品系。
Giant grouper transcriptome was established by using Next Generation Sequencing (NGS) to generate functional genes and microsatellites as Type I markers. More than 20,000 non-redundant Unigenes of giant grouper were identified from 49,722 Unigenes with annotation and classified by GeneOntology (GO). 4701 microsatellite DNA markers including 1779 di-nucleotide (n>8), 1996 tri- (6), 247 tetra- (n>5), 113 penta- (n>4) and 556 hexa-nucleotide (n>3) were identified from 49,722 Unigenes of giant grouper. Among these microsatellites, 2729 SSRs located in the 5’-UTR (495), coding region (1218) and 3’-UTR (862) were classified by enriched GO. From the 73 skeletal myogenesis SSRs and 9 unclassified SSRs, 55 SSRs were successfully screened with 31 SSRs (56%) were found to be polymorphic. The observed (Ho) and expected (He) heterozygosities varied from 0.01 to 0.79 and from 0.01 to 1.0, respectively. Microsatellites in 3’-UTR are more polymorphic (81%) than 5’-UTR (20%) and coding region (21%). 1 deletion marker (Ela8112d) and 9 microsatellites markers (Ela8112a, Ela9156b, Ela28217, Ela22373, Ela28216, Ela25872, Ela348, Ela13962, Ela22361) with different genotypes were found to be significantly associated with the measured growth traits phenotypes (body weight) in fast and slow growth fish. Unexpectedly, a 51 bp deletion was detected within the PCR fragment of (GT)n SSR in 3’-UTR of Ela8112d showing greater heterozygosity. A greater heterozygotes genotype frequency difference was associated with slow growth traits and the true was for the reverse. 5 microsatellite markers (Ela9156b, Ela28216, Ela348, Ela13962, Ela22361) and 1 deletion DNA markers (Ela8112d) associated with fast growth microsatellite markers were validated in a field trial of 96 giant groupers with selected large breed from Mariculture Research Center of Fisheries Research Institutes. In conclusion, 7 microsatellite markers associated with fast growth trait in giant grouper has been established in this study. This finding will set a benchmark for high quality giant grouper breeds.
目  錄
謝  辭 i
摘  要 ii
Abstract iii
壹、前言 1
一、 龍膽石斑簡介 1
二、 傳統育種 2
三、 分子標誌 (Molecular Marker) 3
四、 微衛星DNA (microsatellite DNA) 4
五、 分子標誌輔助育種 (Marker-Assisted Selection) 7
六、 石斑魚的分子標誌 7
七、 研究目的 8
貳、材料與方法 9
一、實驗動物 9
二、實驗材料 10
1. 實驗質體 10
2. 實驗菌株 10
3. 培養基 10
4. 生物及化學藥品 10
5. 市售生物反應套組 11
6. 儀器設備 11
7.引子列表 12
三、實驗方法 14
1. 建立龍膽石斑轉錄體資料庫 14
2. 建立龍膽石斑轉錄體SSR資料庫與偵測SSR marker 16
3. 定序 (Auto- sequencing) 18
參、結果 22
一、龍膽石班轉錄體微衛星DNA資料庫 22
二、骨骼肌生成相關SSR的檢測分析 23
三、SSR分析D79族群成長快速與成長緩慢龍膽石斑各12隻 23
四、SSR分析D79族群成長快速與成長緩慢龍膽石斑 24
五、SSR分析D100成長快速龍膽石斑族群 25
肆、討論 27
伍、結論 33
陸、參考文獻 34
附錄 75
附錄1.1、D79族群10個與體重顯著相關的分子標誌基因型與魚隻基礎資料 75
附錄1.2、D79族群21個有多型性SSR但與體重無顯著相關的基因型與魚隻基礎資料 77
附錄1.3、D79族群24個無多型性SSR的基因型與魚隻基礎資料 83
附錄2.1、D100族群10個與體重顯著相關分子標誌的基因型與魚隻基礎資料 89
附錄2.2、D100族群2個極低多型性SSR與5個有多型性但與體重無相關SSR的基因型與魚隻基礎資料 93
附錄3.1、10個與體重相關分子標誌的等位基因片段鹼基對長度列表 96
附錄3.2、21個有多型性但與體重無顯著相關SSR的等位基因片段鹼基對長度列表 97
附錄3.3、24個無多型性SSR的等位基因片段鹼基對長度列表 99
附錄4、分子標誌技術比較 100

圖 目 錄
圖一、龍膽石斑du-nucleotide microsatellite 分布 50
圖二、10個篩選出的分子標誌等位基因片段訊號示意圖 51
(A) Ela8112d, (B) Ela8112a, (C) Ela9156b, (D) Ela28217, (E) Ela22373, (F) Ela28216, (G) Ela25872, (H) Ela348, (I) Ela13962, (J) Ela22361 51
圖三、D79族群Ela8112d定序結果 53
圖四、(A) D79族群,(B) D100族群中Ela8112d marker的分布情形 54

表 目 錄
表一、D79族群採樣時的基本資料 55
表二、水產試驗所海水繁養殖研究中心蓄養的龍膽石斑採樣時的基本資料 56
表三、龍膽石斑轉錄體符合定義的4701個SSR 57
表四、55個可偵測SSR的多型性,PIC、Ho、He 58
表五、55個可檢測SSR的在D70族群的多型性 60
位於3’ UTR的Ela6645在分析D100族群時出現另一個等位基因,但極低的多型性並不具有深入研究的價值。 60
表六、D79成長快與慢各12隻魚SSR基因型與體重關係關係 61
表七、D79族群的SSR的基因型與體重關係分析 64
表八、D100生長快速族群的10個與體重相關的SSR分析 67
表九、D100族群內基因型與體重ANOVA分析 70
表十、D79與D100族群基因型比較 72
表十一、與體重顯著相關的markers在不同族群比較 74


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