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標題: | 低覆蓋倍率隨機定序開發SSR標誌及其策略探討 SSR marker mining and its strategy under low-coverage shotgun sequencing |
作者: | Yen-Yu Lin 林延諭 |
指導教授: | 胡凱康(Kae-Kang Hwu) |
關鍵字: | 簡單重複序列,微衛星序列,分子標誌,重複序列探勘,焦磷酸定序,低覆蓋倍率, SSR,Microsatellite,Marker mining,Pyrosequencing,Shotgun sequencing,Low-coverage, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64719 |
全文授權: | 有償授權 |
顯示於系所單位: | 農藝學系 |
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