發(fā)布時間：2018-09-17 20:32

【摘要】：黃瓜白粉病是黃瓜(CucumissativusL.)生產(chǎn)上的三大主要病害之一,發(fā)病時不但降低植株的光合效能,同時影響植株產(chǎn)量和果實品質(zhì),發(fā)病嚴重時常常引起30%左右的減產(chǎn)。黃瓜抗白粉病新品種選育及應(yīng)用是克服白粉病危害的根本技術(shù)途徑�；诨蚪M測序技術(shù)和生物信息學(xué)的方法探究抗病基因已成為可能。本研究利用高通量Illumina測序技術(shù),對實驗室多年篩選獲得的一個具有高抗白粉病且能穩(wěn)定遺傳的片段代換系SSL508-28和高感白粉病受體親本D8進行了全基因組重測序,對比黃瓜9930參考基因組信息,在SSL508-28中發(fā)現(xiàn)了 468,616 個單核苷酸多態(tài)性位點(single nucleotide polymorphisms,SNPs)和 67,259小片段插入缺失位點(insertion/deletion,InDel),在D8 中獲得了 537,352 個 SNPs和 91,698個InDels。通過對比SSL508-28與D8基因組,共得到了 15,682個SNPs和6,262個InDels,這些SNPs和InDels趨向于集中分布在五號染色體上�；谝陨辖Y(jié)果,我們對獲得的SNPs和InDels進行了功能注釋,發(fā)現(xiàn)有120個SNPs為非同義(non-synonymous)突變,30個InDels為移碼突變(frameshift mutation),這些非同義突變SNPs和移碼突變InDels分布在94個基因當中。為了進一步驗證94個突變基因?qū)SL508-28抗白粉病表型的貢獻,我們對這94個基因進行了功能分類,其中有5個基因?qū)儆诳共?resistance,R)基因家族中NBS-LRR(Nucleotide binding site-leucine-rich repeats)類,利用 qRT-PCR對這 5 個NBS-LRR基因在D8和SSL508-28中接種白粉菌前后的表達量進行檢測,結(jié)果表明,基因Csa2M435460.1和Csa5M579560.1表達量在SSL508-28接種白粉菌后明顯上調(diào)且在48小時達到表達高峰,而D8接種白粉菌前后表達量沒有明顯變化。為了進一步驗證候選基因與黃瓜抗白粉病密切相關(guān),我們通過qRT-PCR分析Csa2M435460.1和Csa5M579560.1在黃瓜不同抗感白粉病品種中的表達量,結(jié)果顯示,Csa2M43546.1和Csa5M579560.1在抗病品種中的表達量高于感病品種,且在供體親本JIN5-508中兩基因的表達量最高。此外,我們還發(fā)現(xiàn)在SSL508-28中基因Csa2M435460.1和Csa5M579560.1的編碼區(qū)發(fā)生的一個非同義突變使半胱氨酸和纈氨酸均突變成苯丙氨酸,因此推測,基因Csa2M435460.1和Csa5M579560.1是黃瓜抗白粉病相關(guān)基因且發(fā)生在基因Csa2M435460.1和Csa5M579560.1中的非同義突變是導(dǎo)致SSL508-28具有高抗白粉病特性的主要原因之一。
[Abstract]:Cucumber powdery mildew is cucumber (CucumissativusL.) One of the three major diseases in production, not only reduce the photosynthetic efficiency of the plant, but also affect the yield and fruit quality of the plant. When the disease is serious, it often causes about 30% reduction in yield. Breeding and application of new cucumber varieties resistant to powdery mildew are the fundamental technical ways to overcome the harm of powdery mildew. It is possible to explore resistance genes based on genome sequencing and bioinformatics. Using high-throughput Illumina sequencing technique, a fragment replacement line SSL508-28 with high resistance to powdery mildew and highly susceptible powdery mildew receptor parent D8 was sequenced. Comparing with reference genome information of cucumber 9930, 468616 SNP (single nucleotide polymorphisms,SNPs and 67259 insertion/deletion,InDel were found in SSL508-28. 537352 SNPs and 91698 InDels. were obtained in D8. By comparing the genomes of SSL508-28 and D8, a total of 15682 SNPs and 6262 InDels, were obtained. These SNPs and InDels tend to be concentrated on chromosome 5. Based on the above results, we annotated the SNPs and InDels, and found that 120 SNPs were non-synonymous mutations, 30 InDels were frameshift mutations (frameshift mutation), and SNPs and InDels were distributed in 94 genes. To further verify the contribution of 94 mutant genes to the phenotypes of SSL508-28 resistance to powdery mildew, we have classified the 94 genes into functional categories, five of which belong to the NBS-LRR (Nucleotide binding site-leucine-rich repeats class in the resistance,R gene family. The expression of 5 NBS-LRR genes in D8 and SSL508-28 was detected by qRT-PCR. The results showed that the expression of Csa2M435460.1 and Csa5M579560.1 increased significantly after inoculation with SSL508-28 and reached the peak at 48 hours. However, the expression of D8 did not change significantly before and after inoculation with powdery mildew. In order to further verify the close relationship between candidate genes and cucumber powdery mildew resistance, we analyzed the expression of Csa2M435460.1 and Csa5M579560.1 in cucumber cultivars with different resistance to powdery mildew by qRT-PCR. The results showed that the expression of Csa2M43546.1 and Csa5M579560.1 in disease-resistant cultivars was higher than that in susceptible varieties. The expression of two genes was the highest in donor parent JIN5-508. In addition, we also found that a nonsynonymous mutation in the coding region of Csa2M435460.1 and Csa5M579560.1 in SSL508-28 caused both cysteine and valine to mutate into phenylalanine. The non-synonymous mutations of genes Csa2M435460.1 and Csa5M579560.1, which are related to powdery mildew resistance in cucumber and occur in gene Csa2M435460.1 and Csa5M579560.1, are one of the main reasons for the high resistance of SSL508-28 to powdery mildew.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S436.421.12

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