基于全基因组重测序获得的具LRR结构域基因的抗黄瓜白粉病功能鉴定
发布时间：2018-09-17 20:32
【摘要】：黄瓜白粉病是黄瓜(CucumissativusL.)生产上的三大主要病害之一,发病时不但降低植株的光合效能,同时影响植株产量和果实品质,发病严重时常常引起30%左右的减产。黄瓜抗白粉病新品种选育及应用是克服白粉病危害的根本技术途径。基于基因组测序技术和生物信息学的方法探究抗病基因已成为可能。本研究利用高通量Illumina测序技术,对实验室多年筛选获得的一个具有高抗白粉病且能稳定遗传的片段代换系SSL508-28和高感白粉病受体亲本D8进行了全基因组重测序,对比黄瓜9930参考基因组信息,在SSL508-28中发现了 468,616 个单核苷酸多态性位点(single nucleotide polymorphisms,SNPs)和 67,259小片段插入缺失位点(insertion/deletion,InDel),在D8 中获得了 537,352 个 SNPs和 91,698个InDels。通过对比SSL508-28与D8基因组,共得到了 15,682个SNPs和6,262个InDels,这些SNPs和InDels趋向于集中分布在五号染色体上。基于以上结果,我们对获得的SNPs和InDels进行了功能注释,发现有120个SNPs为非同义(non-synonymous)突变,30个InDels为移码突变(frameshift mutation),这些非同义突变SNPs和移码突变InDels分布在94个基因当中。为了进一步验证94个突变基因对SSL508-28抗白粉病表型的贡献,我们对这94个基因进行了功能分类,其中有5个基因属于抗病(resistance,R)基因家族中NBS-LRR(Nucleotide binding site-leucine-rich repeats)类,利用 qRT-PCR对这 5 个NBS-LRR基因在D8和SSL508-28中接种白粉菌前后的表达量进行检测,结果表明,基因Csa2M435460.1和Csa5M579560.1表达量在SSL508-28接种白粉菌后明显上调且在48小时达到表达高峰,而D8接种白粉菌前后表达量没有明显变化。为了进一步验证候选基因与黄瓜抗白粉病密切相关,我们通过qRT-PCR分析Csa2M435460.1和Csa5M579560.1在黄瓜不同抗感白粉病品种中的表达量,结果显示,Csa2M43546.1和Csa5M579560.1在抗病品种中的表达量高于感病品种,且在供体亲本JIN5-508中两基因的表达量最高。此外,我们还发现在SSL508-28中基因Csa2M435460.1和Csa5M579560.1的编码区发生的一个非同义突变使半胱氨酸和缬氨酸均突变成苯丙氨酸,因此推测,基因Csa2M435460.1和Csa5M579560.1是黄瓜抗白粉病相关基因且发生在基因Csa2M435460.1和Csa5M579560.1中的非同义突变是导致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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.421.12
本文编号：2247009
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.421.12
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