請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92165完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉瑞芬 | zh_TW |
| dc.contributor.author | 黃順源 | zh_TW |
| dc.contributor.author | Shun-Yuan Huang | en |
| dc.date.accessioned | 2024-03-07T16:23:09Z | - |
| dc.date.available | 2024-03-08 | - |
| dc.date.copyright | 2024-03-07 | - |
| dc.date.issued | 2016 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | REFERENCE
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92165 | - |
| dc.description.abstract | 為了追蹤田間晚疫病菌的分佈與族群遺傳組成，以利其防治與管理，過去已經開發出數種分子標誌，包括RG57 基因型指紋分析。雖然RG57 是目前歐美地區廣為接受的基因分型方法，但由於涉及南方雜合分析，有需要大量基因體DNA、耗時且步驟繁複等缺點。本研究的主旨為藉由簡單序列重複(SSR)分子標誌建立一套快速、高效率的基因分型流程，以便應用於分析臺灣的晚疫病菌族群與區隔海外的菌系。經初步測試，已建立一套包含12 組SSR 分子標誌的複合式PCR 反應流程，能夠高通量地分析晚疫病菌的基因分型。為評估該分子標誌區別晚疫病菌菌系的能力，目前已完成超過200 個來自臺灣與海外菌株的基因分型。RG57 分析結果顯示，臺灣的晚疫病菌以2010 年為界，US 11.2 亞型已經取代典型的US-11系成為田間主要的基因型。SSR 分型資料顯示，近十年來，不同年分的臺灣晚疫病菌株基因型有顯著分化，說明晚疫病菌族群有隨時間演替的現象。但晚疫病菌並沒有在地理上和寄主上有遺傳分化。為區別本土與海外的晚疫病菌，以包含至少8 種菌系的海外菌株作為參考菌株，分析顯示臺灣的菌株在基因型層次能顯著的與US-8、US-22、US-23 區分，但US-7、US-11 與US-16 參考菌株的基因型與臺灣菌株相近。綜合上述結果，相對於傳統的RG57 分型方法，此套SSR 基因分型方法是一套高效率、適合高通量分析的工具，能夠精準的鑑別晚疫病菌的菌系，以快速提供菌相的遺傳資訊，幫助田間晚疫病菌的監控與防治管理。 | zh_TW |
| dc.description.abstract | ABSTRACT
Late blight caused by Phytophthora infestans is a devastating threat to tomato and potato production worldwide. To facilitate studies of fungicide resistance, population genetics, as well as pathogen dissemination and migration of P. infestans, a variety of molecular markers have been developed, including the RG57 fingerprinting marker. Despite it is widely used, this method has drawbacks of being labor intensive, time consuming, and in need of a large amount of DNA. This study aims to develop a fast, effective genotyping method based on simple sequence repeat (SSR; also known as microsatellites). A one-step multiplex PCR method with 12 SSR markers is applied for high-throughput genotyping of P. infestans. RG57 fingerprinting showed the variant US-11.2 replace the typical US-11, becoming the dominant genotype since 2010. To evaluate whether the SSR markers are effective for distinguishing RG57-based lineages, SSR genotypes of over 200 isolates from Taiwan and overseas were analyzed. The isolates collected from different years showed significant genotypic differentiation, suggesting populations in Taiwan were turnover for the past decade. To identify overseas lineages form local populations, reference isolates including at least 5 lineages were applied. Analysis showed local isolates can be distinguished from US-8, US-22, and US-16 lineages on genotypic level, while US-7, US-11 and US-16 are close to isolates in Taiwan. In summary, compared to traditional RG57 fingerprinting, the SSR markers are effective for genotyping of P. infestans, which may provide important information for monitoring and management of P. infestans in the fields. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-03-07T16:23:09Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-03-07T16:23:09Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | CONTENTS
口試委員會審定書 ...........................................................................................................# 誌謝 ....................................................................................................................................i 中文摘要.......................................................................................................................... ii ABSTRACT .................................................................................................................... iii CONTENTS .....................................................................................................................iv LIST OF FIGURES....................................................................................................... viii LIST OF TABLES.............................................................................................................x Chapter 1 前言............................................................................................................1 1.1 晚疫病的重要性............................................................................................1 1.2 晚疫病菌的生物學特性................................................................................1 1.2.1 分類位階...............................................................................................1 1.2.2 晚疫病菌的生活史...............................................................................2 1.2.3 晚疫病的病徵與流行病學...................................................................4 1.3 晚疫病菌的基因分型....................................................................................5 1.3.1 分子標誌...............................................................................................5 1.3.2 異構酶(isozymes) ...............................................................................6 1.3.3 粒線體限制片段長度多型性(mitochondrial restriction fragment length polymorphism; mt-RFLP) ..........................................................6 1.3.4 RG57 指紋分析(RG57 fingerprinting)...............................................7 1.3.5 簡單序列重複(simple sequence repeats; SSR) ..................................8 1.3.6 單核苷酸多型性(single-nucleotide polymorphism, SNP)...............10 1.4 晚疫病菌的族群研究..................................................................................10 1.4.1 愛爾蘭大饑荒晚疫病菌.....................................................................10 1.4.2 晚疫病的起源地爭論.........................................................................11 1.4.3 歐洲晚疫病發生情形與族群研究.....................................................13 1.4.4 北美洲晚疫病發生情形與族群研究.................................................15 1.4.5 中國晚疫病發生情形與族群研究.....................................................17 1.4.6 臺灣的晚疫病發生情形與族群研究.................................................18 Chapter 2 材料方法..................................................................................................20 2.1 晚疫病菌株來源與培養..............................................................................20 2.2 DNA 純化.....................................................................................................20 2.3 RG57 指紋分析............................................................................................21 2.4 SSR 專一性引子對設計..............................................................................22 2.5 多重PCR......................................................................................................23 2.6 複合式PCR..................................................................................................23 2.6.1 毛細管電泳圖譜判讀.........................................................................24 2.6.2 建立基因型資料.................................................................................25 2.6.3 個別基因座基因型統計.....................................................................25 2.6.4 連鎖不平衡(linkage disequilibrium) 檢定......................................26 2.6.5 貝氏分群分析(Bayesian clustering).................................................26 2.6.6 區別成份分析(Discriminant analysis of principle component, DAPC) .............................................................................................................27 2.6.7 複基因座基因型(Multi-locus genotype, MLG) 分析.....................27 2.6.8 計算遺傳距離與繪製親緣樹.............................................................28 2.6.9 分子變異度分析(analysis of molecular variance, AMOVA) ...........29 Chapter 3 結果..........................................................................................................30 3.1 RG57 指紋分析............................................................................................30 3.2 開發SSR 分子標誌.....................................................................................30 3.3 臺灣晚疫病菌的SSR 基因分型.................................................................31 3.4 臺灣晚疫病族群動態分析..........................................................................32 3.4.1 連鎖不平衡(Linkage disequilibrium) 檢定.....................................32 3.4.2 貝氏分群分析.....................................................................................33 3.4.3 晚疫病菌基因型與採集年份的關聯性.............................................33 3.4.4 晚疫病菌基因型與採集地點的關聯性.............................................36 3.4.5 晚疫病菌基因型與寄主的關聯性.....................................................37 3.5 以SSR 分子標誌區分不同菌系的晚疫病菌.............................................37 3.5.1 美國參考菌株(Reference isolates) 的基因分型.............................37 3.5.2 中國參考菌株的基因分型.................................................................38 3.5.3 貝氏分群分析.....................................................................................38 3.5.4 區別成份分析.....................................................................................39 3.5.5 親緣系譜分析.....................................................................................39 Chapter 4 討論..........................................................................................................40 4.1 晚疫病菌US-11 菌系仍然是臺灣的優勢菌系..........................................40 4.2 臺灣的晚疫病菌族群有演替的現象..........................................................41 4.3 族群演替的機制探討..................................................................................42 4.3.1 變異與突變.........................................................................................42 4.3.2 有性生殖.............................................................................................44 4.3.3 有絲分裂重組(mitosis recombination) ............................................45 4.3.4 多倍體或非整倍體.............................................................................45 4.3.5 遺傳漂變(gene drift).........................................................................46 4.3.6 遷徙.....................................................................................................47 4.4 應用SSR 分子標誌高效分析晚疫病菌基因型.........................................47 REFERENCE ..................................................................................................................51 LIST OF FIGURES 圖一、臺灣晚疫病菌株(2005-2013) 的 RG57 指紋分析圖譜。…………………...95 圖二、臺灣晚疫病菌株 (2012-2013) 的 RG57 指紋分析圖譜。………………......96 圖三、臺灣晚疫病菌株(2015 年) 的 RG57 指紋分析圖譜。………………………..97 圖四、應用本研究開發的SSR 分子標誌建立晚疫病菌的親緣系譜。………….....98 圖五、連鎖不平衡檢定顯示臺灣晚疫病菌為無性族群。…………………..............99 圖六、基因座配對連鎖相關性指數顯示Pi4B 與D13 之間的連鎖程度強。……..100 圖七、除去D13 與Pi4B，連鎖不平衡檢定仍顯示臺灣晚疫病菌為無性族群。…101 圖八、貝氏分群分析顯示臺灣的晚疫病菌族群有限度的分化。….……………...102 圖九、區別成份分析顯示不同年分的晚疫病菌株有基因型差異。…...………….103 圖十、發生於2001、2002 與2014 年的晚疫病菌族群MLG 相對豐度較高。.....105 圖十一、不同年份臺灣晚疫病菌族群的MLG 組成。………………………….....106 圖十二、MLG 的遺傳距離與發生年份。...………………………………………....107 圖十三、MLG 發生年份與基於Bruvo 氏遺傳距離的最小生成網絡。………......109 圖十四、MLG 發生年份與基於Provesti 氏遺傳距離的最小生成網絡。………....110 圖十五、MLG 發生年份與基於Nei 氏遺傳距離的最小生成網絡。……………...111 圖十六、AMOVA 顯示不同年份的族群間有顯著分化。……………………….....112 圖十七、區別成份分析顯示不同地理區的族群有沒有顯著的遺傳差異。…........114 圖十八、不同地理區的臺灣晚疫病菌族群其相對豐度相近。…….……………...116 圖十九、不同地理區臺灣晚疫病菌族群的MLG 組成。..………………………...117 圖二十、MLG 來源地理區與基於Bruvo 氏遺傳距離的最小生成網絡。………...119 圖二十一、MLG 來源地理區與基於Provesti 氏遺傳距離的最小生成網絡。…...120 圖二十二、MLG 來源地理區與基於Nei 氏遺傳距離的最小生成網絡。…..........121 圖二十三、AMOVA 顯示不同地理區的族群間沒有顯著分化。….……………...122 圖二十四、已無性校正的AMOVA 顯示不同地理區的族群間沒有顯著分化。…123 圖二十五、區別成份分析顯示不同寄主上的晚疫病菌沒有顯著的遺傳差異。….124 圖二十六、不同寄主的臺灣晚疫病菌族群其相對豐度相近。…….……………...125 圖二十七、不同寄主上臺灣晚疫病菌族群的MLG 組成。………………..............126 圖二十八、MLG 來源寄主與基於Bruvo 氏遺傳距離的最小生成網絡。………….127 圖二十九、MLG 來源寄主與基於Provesti 氏遺傳距離的最小生成網絡。...........128 圖三十、MLG 來源寄主與基於Nei 氏遺傳距離的最小生成網絡。…..................129 圖三十一、貝氏分群分析顯示209 個晚疫病菌菌株能區分為三群。……….…...130 圖三十二、區別成份分析顯示臺灣菌株與許多參考菌株有顯著的遺傳差異。…...132 圖三十三、親緣系譜顯示臺灣菌株位於與許多參考菌株不同的分支上。….…...133 圖三十四、US-7、US-11、US-16 參考菌株位在臺灣菌株的親緣網絡中。..........135 LIST OF TABLES 表一、本研究所使用的臺灣晚疫病菌株………………………………………….62 表二、本研究使用的中國晚疫病菌株………………………………………….68 表三、本研究所使用的引子………………………………………………………….70 表四、本研究開發出十組有較佳多型性的SSR 分子標誌…………………….72 表五、應用本研究開發的SSR 分子標誌分析臺灣、中國與美國菌株………….74 表六、應用本研究開發的SSR 分子標誌分析臺灣的晚疫病菌菌株………….75 表七、臺灣晚疫病菌菌株(1998-2015) 之 SSR 基因型統計…………………….76 表八、臺灣晚疫病菌菌株(1992-1998) 之 SSR 基因型統計…………………….78 表九、臺灣晚疫病菌菌株可歸類為60 個不同的複基因座基因型(MLG) …….80 表十、不同年份的臺灣晚疫病菌菌株多樣性指數……………………………….83 表十一、AMOVA 分析顯示不同年份的晚疫病菌菌株間有輕度到中度分化….....85 表十二、個別基因座ϕ st 分化指數分析不同年分的臺灣晚疫病菌株………….86 表十三、來自不同地理區的臺灣晚疫病菌株多樣性指數…………………….87 表十四、AMOVA 分析顯示來自不同地理區的臺灣晚疫病菌沒有顯著的分化.88 表十五、來自不同寄主的臺灣晚疫病菌株多樣性指數………………………….89 表十六、AMOVA 分析顯示來自不同寄主的臺灣晚疫病菌沒有顯著分化…….90 表十七、美國參考菌株的基因型……………………………………………….91 表十八、中國參考菌株的基因型……………………………………………….92 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 晚疫病菌 | zh_TW |
| dc.subject | 基因分型 | zh_TW |
| dc.subject | 簡單序列重複 | zh_TW |
| dc.subject | RG57 | zh_TW |
| dc.subject | RG57 | en |
| dc.subject | Phytopthora infestans | en |
| dc.subject | genotyping | en |
| dc.subject | SSR | en |
| dc.title | 建立 SSR 分子標誌及分析臺灣晚疫病菌族群遺傳動態 | zh_TW |
| dc.title | Development of microsatellite markers and genetic assessment for population dynamics of Phytophthora infestans from Taiwan | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 104-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 楊景程;安寶貞;鍾嘉綾 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | 晚疫病菌,基因分型,簡單序列重複,RG57, | zh_TW |
| dc.subject.keyword | Phytopthora infestans,genotyping,SSR,RG57, | en |
| dc.relation.page | 135 | - |
| dc.identifier.doi | 10.6342/NTU201602731 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2016-08-18 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 植物病理與微生物學系 | - |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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| ntu-104-2.pdf 未授權公開取用 | 4.48 MB | Adobe PDF |
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