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標題: | 應用集群分離RNA測序分析 研究臺灣大豆「高雄11號」之白粉病抗病基因座 Application of Bulk Segregant RNA-Seq Analysis on the Powdery Mildew Resistance Loci in Soybean Cultivar 'KH11' of Taiwan |
作者: | 黃承濬 Cheng-Chun Huang |
指導教授: | 張皓巽 Hao-Xun Chang |
關鍵字: | 大豆,白粉病,抗病基因座,差異表現基因分析,集群分離分析, Allele-specific PCR,Bulk-segregant RNA-Seq,Differentially expressed gene,Soybean,Powdery mildew,Resistance, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 大豆白粉病是重要的大豆病害之一,而使用抗病品種已被證實可以有效降低大豆白粉病所造成的危害。以往其他國家針對大豆白粉病抗病的研究多導向在大豆16號染色體上的Rmd 基因座,而其他地區的大豆品種是否有其他抗性基因仍有待調查。本研究利用群聚分離分析法定位台灣大豆品種「高雄11號」上之抗病基因座。利用比較抗病親本「高雄11號」和感病親本「花蓮1號」、以及抗病子代和感病子代的RNA序列,找出與抗白粉病相關的基因座可能位於第 6、15及16條染色體上。而使用HISAT2和Kallisto兩種方法分析抗感病相關差異表現基因,總共發現1687個差異表現基因在兩種方法間的分析上都被認為與白粉病抗性高度相關。另根據連鎖不平衡區間分析,在第6、15及16條染色體上選擇單核苷酸多態性以設計等位基因專一性引子對。結果發現第6及15條染色體的等位基因專一性引子對無法區分抗感病基因型,唯有第16條染色體上的單核苷酸多態性可區分抗感病基因型,且確率達為82%,與前人文獻提及的SSR引子對Satt431相近。研究結果發現台灣大豆品種「高雄11號」上抗白粉病抗性主要由第16號染色體所調控。本篇研究亦設計了可以預測「高雄11號」抗病表現型之等位基因專一性引子對,期許可以應用於「高雄11號」作為親本之大豆抗病品種的育成。 Powdery mildew (PM) is one of the important soybean (Glycine max) diseases worldwide, and the implementation of soybean resistance has shown to be effective in managing PM. To date, there is only one PM resistance QTL, the Rmd locus identified on soybean chromosome (Chr) 16 based on studying the varieties from the United States and China. Whether additional PM resistance existed in soybean varieties from other geographic origins remains to be answered. This project focuses on the PM-resistant variety 'Kaohsiung 11' ('KH11') of Taiwan, and an F3 population was generated by crossing 'KH11' with the PM-susceptible variety 'Hualien 1' ('HL1'). Bulk segregant RNA-Seq (BSR-Seq) was applied to capture the gene expression and genotypic variation between the resistant bulk (R bulk) and the susceptible bulk (S bulk), as well as the parents. The ∆(SNP-index) and G' value analyses identified significant SNPs and Indels on Chr06, Chr15, and Chr16 associated with PM resistance. To validate the differential expression of genes (DEGs), consensus 1687 DEGs identified by both HISAT2 and Kallisto methods were considered as candidate genes associated with PM resistance. The candidate genes on Chr06, Chr15, and Chr16 were selected based on the Log2 fold change value and the relative position with the significant SNPs, and primers that can be used in the allele-specific polymerase chain reaction (AS-PCR) were designed. While the AS-PCR primers designed for Chr06 or Chr15 cannot distinguish the resistant and susceptible genotypes, AS-PCR primers on Chr16 were successful with an 82% accuracy rate, similar to the simple sequence repeat (SSR) marker Satt431 from previous studies. These results not only confirm the resistant source in the Taiwanese soybean cultivar 'KH11' is predominantly govern by the Rmd locus on Chr16, but also suggest the ASPs on Chr16 designed in this study can be used as a molecular marker for breeding soybean PM resistance derived from 'KH11'. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91237 |
DOI: | 10.6342/NTU202304354 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 植物醫學碩士學位學程 |
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