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研究生:張為斌
研究生(外文):Wei-Bin Chang
論文名稱:臺農84號抗稻熱病基因座之分析定位
論文名稱(外文):Identification of quantitative trait loci for blast resistance in Taiwan rice variety Tainung 84
指導教授:鍾嘉綾
口試委員:沈偉強董致韡吳志文
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:126
中文關鍵詞:數量性狀基因座定位生理小種判別品種抗病遺傳
外文關鍵詞:QTL mappingMagnaporthe oryzaephysiological racesdifferential linesresistance genetics
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由Magnaporthe oryzae所引起的稻熱病,是全球稻米生產之重要威脅,目前主要以種植抗病品種,搭配合理化施肥及化學防治進行管理。臺灣雖然陸續有抗稻熱病品種的育成,但由於其所帶抗病基因/基因座不明,加上田間稻熱病菌的生理小種組成複雜且變異頻繁,抗病品種往往在大面積推廣種植數年後即失去抗性。本研究以數量性狀基因座 (quantitative trait loci, QTL) 定位法,針對具有優良抗稻熱病特性之新育成品種「臺農84號」 (Tainung 84, TNG84) 及近年臺灣栽培最廣之感病品種「臺南11號」 (Tainan 11, TN11) 之雜交後代,尋找調控抗性之基因座。本研究使用分離自臺灣田間、分屬不同Pot2分群、不同生理小種之三株稻熱病菌菌株D41-2、12CY-MS1-2及12IL-TT4-1進行F2及F2:3水稻族群稻熱病接種實驗,並運用genotyping by sequencing (GBS) 技術針對122株F2子代進行基因型鑑定,獲得7,742個單一核苷酸多型性 (single-nucleotide polymorphism, SNPs) 位點。結果顯示,TNG84對前述三株菌株的抗病因子,為一個位於第12條染色體約52 cM位置 (physical map position: 9,547,082-15,160,145 bp on IRGSP 1.0 map) 之主效QTL,其 logarithm of odds (LOD) score為20.553-31.687,R2為54.3-69.8%。進一步利用D41-2接種來自16個F2:3之665株F2:4個體,並設計5個切割擴增多型性序列 (cleaved amplified polymorphic sequences, CAPS) 分子標誌確認植株基因型,將QTL限縮至10,012,254-13,020,487 bp區間內。根據該區間內候選基因之序列多型性及不同菌株接種結果,判斷TNG84之抗性可能同時來自已被選殖之Pita基因及鄰近抗病基因。

Rice blast, caused by Magnaporthe oryzae, is a serious constraint to rice production. The use of resistance varieties, in combination with proper fertilizer management and pesticide application, is a widely adopted strategy for effective control of this disease. Through years, breeders in Taiwan have developed several cultivars possessing high level of blast resistance. However, mechanisms underlying the resistance remain unknown. Besides, the resistance can often be overcome after a few years of widespread cultivation. This is mainly due to the complex genetic diversity of M. oryzae populations and their high mutation rates at avirulence loci. This study aimed to uncover the resistance/susceptibility genetics of “Tainung 84” (TNG84, a resistant cultivar released in 2010) and “Tainan 11” (TN11, a most widely cultivated rice varirty in Taiwan, moderately susceptible to blast) using quantitative trait loci (QTL) mapping approach. Three field isolates of M. oryzae belonging to different Pot2 groups and physiological races, D41-2, 12CY-MS1-2, and 12IL-TT4-1, were used to inoculate the F2 and F2:3 progeny from “TN11 x TNG84”. A total of 7,742 single-nucleotide polymorphism (SNPs) were identified for 122 F2 individuals by using genotyping by sequencing (GBS) analysis. Composite interval mapping revealed that TNG84 harbors a major-effect QTL conferring resistance to all the three isolates. The QTL was located at ~52 cM on chromosome 12 (physical map position: 9,547,082-15,160,145 bp on IRGSP 1.0 map, logarithm of odds score: 20.553-31.687, R2 = 54.3-69.8%). A population consisting of 665 F2:4 individuals from 16 F2:3 families was phenotyped by inoculation with D41-2 and genotyped by five newly-developed cleaved amplified polymorphic sequences (CAPS) markers. Trait-marker association delineated the major QTL to a region between 10,012,254-13,020,487 bp. Sequence variations of several candidate genes in the region, together with the reaction patterns of different M. oryzae isolates, suggested that the superior resistance of TNG84 is likely conferred by the previously-cloned Pita gene and another gene (s) in the neighboring region.

誌謝 i
中文摘要 ii
Abstract iii
目錄 iv
表目錄 vi
圖目錄 vii
縮寫對照表 viii
壹、前言 9
貳、前人研究 12
2.1 稻熱病菌生理小種 12
2.2 水稻對稻熱病之抗性遺傳 15
2.3 數量性狀基因座 (quantitative trait loci, QTL) 分析 17
2.4 高通量基因型檢定方法 20
參、材料與方法 23
3.1 水稻親本及遺傳定位族群 23
3.2 表現型鑑定 24
3.2.1 稻熱病菌來源、培養方法、保存 24
3.2.2 水稻幼苗栽培 26
3.2.3 稻熱病接種試驗 26
3.2.4 稻熱病罹病程度調查 28
3.2.5 族群分離率卡方檢定分析 29
3.3 基因型分析 30
3.3.1 水稻基因體DNA之萃取 30
3.3.2 NGS (next-generation sequencing) 資料與分析 31
3.3.3 基因圖譜SNP分析 31
3.3.4 F2族群之基因型分析 32
3.3.5 GBS資料分析 34
3.3.6 QTL資料與分析 35
3.3.7 候選片段基因型分析 36
3.3.8 切割擴增多型性序列分子標誌 (cleaved amplified polymorphic sequences markers, CAPS markers) 設計 36
3.3.9 F2:4族群之分析 38
肆、結果 39
4.1 稻熱病菌分子分群、病原型分析 39
4.2 稻熱病菌F2接種試驗 39
4.3 稻熱病菌F2:3接種試驗 40
4.4 NGS資料分析結果 41
4.5 GBS資料分析結果 42
4.6 臺南11號與臺農84號F2雜交族群連鎖圖譜 44
4.7 臺農84號抗稻熱病基因座定位 44
4.8 F2:4族群分析結果 45
4.9 候選片段候選基因分析 47
伍、討論 49
陸、圖表 60
柒、參考文獻 109
捌、附錄 117


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