本文選題：葡萄　切入點(diǎn)：白腐病　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：白腐病是葡萄主要病害之一,是由白腐病原菌(Coniothyrium diplodiella(Speq.)Sacc)引起的。多數(shù)栽培葡萄品種易感染白腐病,葡萄屬野生種是抗白腐病的重要種質(zhì)資源,其中起源于我國的山葡萄(Va.armuRENSIS Rupr.)'雙優(yōu)'具有抗白腐病特性。本研究以'紅地球'×'雙優(yōu)'的雜交后代群體作為試驗(yàn)材料,在本試驗(yàn)室已構(gòu)建的葡萄分子遺傳連鎖圖譜上,開展葡萄白腐病的抗性鑒定,并進(jìn)行白腐病抗性的QTL定位,最終對(duì)抗白腐病候選基因進(jìn)行熒光定量PCR分析。主要研究結(jié)果如下:利用rDNA-ITS序列分析技術(shù)分析培養(yǎng)的供試菌株,將獲得的ITS序列與GenBank中知序列進(jìn)行Blast比對(duì),并利用MEGA4.1軟件進(jìn)行ITS序列聚類分析。本試驗(yàn)培養(yǎng)所得菌株為葡萄白腐菌,可以開展后續(xù)白腐病原菌侵染離體葉片試驗(yàn)。連續(xù)兩年采用室內(nèi)離體葉片鑒定的方法,對(duì)'紅地球'×'雙優(yōu)'雜交產(chǎn)生的149株F1代群體開展白腐病抗性評(píng)價(jià)。試驗(yàn)群體單株發(fā)病情況存在明顯差異,后代群體頻率分布呈現(xiàn)出單峰分布,遺傳特征表現(xiàn)為連續(xù)分布,符合數(shù)量性狀遺傳的規(guī)律,抗性評(píng)價(jià)表型值可用于后續(xù)的白腐病QTL定位。結(jié)合試驗(yàn)室前期構(gòu)建的'紅地球'×'雙優(yōu)'分子遺傳圖譜,應(yīng)用MapQTL 5.0對(duì)葡萄白腐病抗性進(jìn)行QTL定位,共檢測(cè)到17個(gè)與白腐病抗性相關(guān)的位點(diǎn)。在雙親整合圖譜上共檢測(cè)到6個(gè)QTLs,定位在LG1、LG7、LG11和LG15連鎖群上;在母本'紅地球'遺傳連鎖圖譜上共檢測(cè)到5個(gè)QTLs,定位在LG1、LG7、LG11和LG15連鎖群上;在父本'雙優(yōu)'遺傳連鎖圖譜上共檢測(cè)到6個(gè)QTLs,定位在LG1、LG7、LG11和LG16連鎖群上。在白腐病抗性QTL初定位結(jié)果的基礎(chǔ)上,對(duì)定位區(qū)段內(nèi)的基因進(jìn)行生物信息學(xué)分析,篩選出抗病候選基因7IFY6B,應(yīng)用熒光定量PCR技術(shù)分析基因在抗病和感病葡萄葉片中的表達(dá)。無論在葡萄抗病或感病葡萄葉片中,TIFY6B基因都受到了白腐菌的誘導(dǎo),說明TIFY6B基因可能為潛在的葡萄抗白腐病基因,為分子標(biāo)記輔助選擇育種奠定理論基礎(chǔ)。
[Abstract]:White rot is one of the main diseases of grape, which is caused by Coniothyrium diplodiella.Speq.Sacc. most cultivated grape varieties are susceptible to white rot, and wild grape species are important germplasm resources against white rot. Va.armuRENSIS Rupr.'Shuangyou', which originated from China, has the characteristics of resistance to white rot. In this study, the hybrid progenies of 'Red Earth' 脳 'Shuangyou' were used as experimental materials on the molecular genetic linkage map of grape, which has been constructed in our laboratory. The identification of resistance to white rot of grape was carried out, and the QTL mapping of resistance to white rot was carried out. Finally, fluorescent quantitative PCR analysis of candidate genes against white rot was carried out. The main results were as follows: the tested strains were analyzed by rDNA-ITS sequence analysis technique. The obtained ITS sequence was compared with the known GenBank sequence by Blast, and the ITS sequence cluster analysis was carried out by using MEGA4.1 software. The strain cultured in this experiment was white rot fungus of grape. It is possible to carry out subsequent experiments on the infection of white rot pathogens against leaves in vitro. For two consecutive years, the method of identification of isolated leaves in laboratory was used. White rot resistance was evaluated in 149 F1 populations from 'Red Earth' 脳 'double you' hybrids. There were significant differences in the incidence of single plant in the tested populations. The frequency distribution of progenies showed a single peak distribution, and the genetic characteristics showed a continuous distribution. In accordance with the law of inheritance of quantitative traits, the evaluation of resistance to white rot can be used for the subsequent QTL mapping of white rot. Combined with the molecular genetic map of 'Red Earth' 脳 'double you' constructed in the early stage of laboratory, MapQTL 5.0 was used to map the resistance to white rot of grape by QTL. A total of 17 loci associated with white rot resistance were detected. Six QTLs were detected on the amphiphilic integration map, which were located in the LG1LLG7LG11 and LG15 linkage groups, and 5 QTLswere detected on the genetic linkage map of the female parent 'Red Earth', which were located in the LG1LL7LG11 and LG15 linkage groups. A total of 6 QTLs were detected on the genetic linkage map of male parent 'Shuangyou', located in LG1 / LG7 / LG11 and LG16 linkage groups. Based on the results of QTL initial mapping of resistance to white rot, bioinformatics analysis was carried out on the genes in the locus. The candidate gene 7IFY6B was screened, and the expression of 7IFY6B gene in disease-resistant and disease-susceptible grape leaves was analyzed by fluorescence quantitative PCR. The TIFY6B gene was induced by white rot fungus in both disease-resistant and disease-susceptible grape leaves. The results suggested that TIFY6B gene might be a potential gene for resistance to white rot in grape and lay a theoretical foundation for molecular marker-assisted selection breeding.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.631

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