臺灣博碩士論文加值系統

簡單重複序列 (simple sequence repeat, SSR) 標誌因具有共顯性、易操作、多對偶基因、在基因體中含量豐富等特性而被廣泛的應用在各種遺傳研究中。近年來雖有許多研究致力於開發番椒的簡單重複序列標誌，但其數量仍不足以建構飽和的連鎖圖譜。本研究利用454焦磷酸定序系統將番椒 (Capsicum annuum) 進行全基因組隨機定序，透過自行撰寫的程式串連Tandem Repeats Finder (TRF)與Primer3資料，快速開發大量番椒簡單重複序列標誌，並以15個 C. annuum 與1個Capsicum baccatum番椒品種進行120組引子之驗證。驗證結果共有104組引子 (87%) 可產生正常的擴增產物，65組引子 (54%) 在15個C. annuum品種內具有多型性，不論是擴增成功率或多型性比例皆高於已公開標誌。為探討最佳的開發策略，進一步利用Flowsim以水稻參考序列產生454焦磷酸定序資料，對簡單重複序列標誌開發過程之序列組裝、探勘工具及引子篩選方式進行系統性的比較。模擬結果顯示以低覆蓋倍率的全基因組隨機定序資料進行簡單重複序列標誌開發時，無需先組裝序列，因為低覆蓋倍率的序列組裝無法有效延長序列增加引子數量，並且因導入錯誤序列資訊，造成無法成功擴增的引子比例增加。與常用的Msatcommander與QDD兩探勘工具相比，TRF可探勘到最多簡單重複序列；TRF結合reverse e-PCR或BLAST等工具，以引子序列對定序結果進行比對，排除在2條以上序列中具有黏合位置的引子組，可進一步減少具有多個擴增產物的引子比例。基於本研究之發現，標誌開發者可擬定更正確且有效率的標誌開發策略，並減少後續引子驗證成本，以低覆蓋倍率的全基因組隨機定序快速開發大量簡單重複序列標誌。

Simple sequence repeat (SSR) markers have been widely used in many areas of genetic research due to their codominance, ease of use, multiallelic nature, and abundance characteristics in the genome. Though various SSR markers in pepper have been published, it is still not sufficient to create a highly saturated linkage map. Here we utilized the 454 pyrosequencing technique to perform whole genome shotgun sequencing for pepper (Capsicum annuum). Tandem Repeats Finder (TRF) was applied to identify SSRs followed by primer design using Primer3. A Perl script was written to connect these processes. A total of 120 designed primers were verified by 15 varieties of C. annuum and 1 variety of C. baccatum. Verified results show that 104 primer sets were successfully amplified (87%), among them, 65 primer sets possess high polymorphism between the 15 varieties of C. annuum. The success rate and polymorphism of these designed primers were higher than the previously published primers. In order to determine an optimum strategy for SSR marker development, simulated shotgun sequence reads were generated using Flowsim based on rice reference genome sequences to compare the effects of assembly, mining tools, and primer selected methods on SSR primer development. Simulation results revealed that assembling low coverage genomic shotgun sequences not only did not elongate the sequence effectively to facilitate primer design, but also may introduce false sequence information. Compared with commonly used mining tools such as Msatcommander and QDD, we obtained the highest number of SSRs by TRF. We also found that using TRF, coupling with re-PCR or BLAST against original sequence reads may effectively eliminate potentially redundant markers while maintaining higher marker yield. Our findings may provide researchers an efficient SSR marker development strategy and decrease further primer verification cost.

目錄
致謝 i
中文摘要 ii
ABSTRACT iii
目錄 iv
圖目錄 vi
表目錄 vii
對照表 viii
第一章 前言 1
第二章 番椒SSR標誌開發 3
2.1 前言 3
2.2 前人研究 4
2.3 材料與方法 6
2.3.1 試驗材料 6
2.3.2 核DNA抽取方法 6
2.3.3 樣品葉片DNA萃取、定量及品質檢定 7
2.3.4 序列修剪與組裝 8
2.3.5 探勘簡單重複序列 8
2.3.6 簡單重複序列的分類方式 9
2.3.7 引子設計、挑選及合成 10
2.3.8 SSR multiplex-ready PCR 11
2.3.9 毛細管電泳分析 11
2.3.10 螢光資料判讀 11
2.4 結果 14
2.4.1 SSR探勘與引子設計 14
2.4.2 引子評估結果 15
2.5 討論 24
2.5.1 開發效率：成功率與多型性比例 24
2.5.2 SSR開發方式 24
2.5.3 序列組裝與引子篩選 25
2.5.4 探勘工具 26
第三章 以模擬尋找最佳開發策略 29
3.1 前言 29
3.2 前人研究 30
3.2.1 454定序平台原理 30
3.2.2 模擬工具 31
3.2.3 探勘工具 32
3.2.4 重複序列與多型性間的關係 34
3.3 材料與方法 35
3.3.1 電腦硬體設備 35
3.3.2 以Flowsim產生模擬資料 35
3.3.3 序列修剪 35
3.3.4 序列組裝與mapping 36
3.3.5 探勘SSR 36
3.3.6 引子可用性篩選 37
3.3.7 引子驗證方法 39
3.4 結果與討論 41
3.4.1 序列組裝 41
3.4.2 探勘工具之探勘效率比較 43
3.4.3 增加多型性限制條件 44
3.4.4 建議開發程序 45
3.4.5 重新探勘番椒SSR進行實際驗證 45
3.4.6 應用與展望 47
第四章 結論 57
參考文獻 59
附錄 63

圖目錄
圖1、番椒NGS定序長度與序列修剪前後之分布 20
圖2、各組裝倍率發生次數之分布圖 20
圖3、組裝倍率示意圖 21
圖4、SSR序列探勘結果 21
圖5、各種SSR重複單位的數量 22
圖6、SSR序列長度分布 23
圖7、引子驗證示意圖 40
圖8、不同定序量組裝前後之引子總數 50
圖9、序列組裝造成引子類別數量的改變 50
圖10、刪除高組裝倍率疊連群的影響 51
圖11、刪除低組裝倍率連群的影響 51
圖12、篩選條件與探勘軟體對引子類別的影響 52
圖13、增加多型性限制條件後對引子類別的影響 53
圖14、番椒重新探勘SSR結果 53
圖15、番椒重新探勘SSR後各種重複單位的數量 54
圖16、番椒重新探勘後各種SSR重複單位之重複次數分布 55
圖17、架設於臺大農藝系伺服器上的Galaxy平臺擷圖 56
圖18、不同模擬參數在序列修剪後序列長度分布改變 68
圖19、番椒序列修剪前後品質分數的改變 69
圖20、不同模擬參數在序列修剪後品質分布改變 70

表目錄
表1、番椒種原資料 13
表2、序列修剪對序列數量的影響 16
表3、序列組裝摘要 16
表4、SSR探勘結果，SSR序列數量與可設計引子的序列數量 17
表5、引子擴增狀態 18
表6、引子多型性狀態 19
表7、序列組裝對序列數量及長度的影響 48
表8、番椒SSR重新探勘結果 49
表9、不同模擬條件之比較 67

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