芥菜型油菜叶绿体基因组DNA多态性研究

发布时间：2018-03-18 20:18

本文选题：芥菜型油菜　切入点：Illumina测序　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：芥菜型油菜是十字花科芸薹属的异源四倍体种,具有抗旱、抗寒、耐贫瘠等特点;主要分布于我国的西部地区,栽培历史悠久,资源丰富。叶绿体基因组因其分子量小、非重组性、高度保守性、单亲遗传等特点,在揭示物种起源进化、阐述生物多样性等方面意义重大。基于叶绿体基因组高度保守的特性,在具备有效参考序列的前提下,本研究采用Illumina测序技术,对来源广泛的芥菜型油菜材料进行高通量测序,用Sanger测序验证变异频率的可靠性,并筛选部分基因对其多态性进行研究,所获结果如下:1、对叶绿体的分离方法及条件进行优化,以大规模提取DNA。主要对以下操作进行了优化:在叶绿体的分离过程中增加了200 g离心2 min的操作,该操作可以离掉细胞壁碎片和细胞核颗粒,省去了用percoll梯度离心。还用DTT替代了与它作用相似的β-巯基乙醇,刺激性气味更小、毒性更低,该方法操作简单,既降低了实验成本,又提高了纯度和产量。2、Illumina测序技术发掘叶绿体基因组DNA的多态性。通过对1549份芥菜型油菜材料的叶绿体基因组进行混样测序(每50份为一个混样池,共31个),在2%的变异频率之上,共找到变异位点573个,其中位于基因间隔区的有199个,基因上的有374个,且这374个变异位点集中于31个叶绿体基因。3、利用Sanger测序技术验证Illumina测序结果的可靠性。通过对变异频率为2%-3%、6%-7%、20%、4%-23%、8%、10%、12%-15%等不同变异频率分别筛选合适的区段进行Sanger测序验证,发现Illumina测序结果的可靠性在12%以上;淘汰了诸多不可靠的变异频率对应的的变异位点,为后续的研究奠定了基础,也为后来的研究提供了参考。4、叶绿体基因rps19及一个基因间隔区的多态性分析。利用Sanger测序技术从不同省份筛选的96份材料中,rps19基因只在来自英国的材料1490中有4个变异位点,可能在长期的地理隔离中发生了进化。在基因间隔区,有52份材料具有两个相同的变异位点,分别为位点64298,A变为T,以及位点64767,A变为C;另有一个来自瑞典的材料887显示较多的30个变异位点。由此可见,相对于功能基因,基因间隔区进化速率相对较快,地域差异可能也是造成DNA多态性的原因之一。
[Abstract]:Mustard rape is an allotetraploid species of Brassica in the cruciferous family. It has the characteristics of drought resistance, cold resistance and barren tolerance. It is mainly distributed in the western part of China with a long cultivation history and rich resources. The chloroplast genome is small because of its small molecular weight. The characteristics of non-recombination, highly conservative and single parent inheritance are of great significance in revealing the evolution of species origin and biodiversity. Based on the highly conserved nature of chloroplast genome and the availability of effective reference sequences, In this study, high-throughput sequencing of mustard rape was carried out by using Illumina sequencing technique. The reliability of mutation frequency was verified by Sanger sequencing, and some genes were screened to study its polymorphism. The results are as follows: 1. The separation methods and conditions of chloroplast were optimized for large-scale extraction of DNA. the following operations were optimized: 200g centrifugation 2 min was added to the separation of chloroplast. This procedure can remove cell wall fragments and nuclear particles, and obviate the use of percoll gradient centrifugation. DTT is also used to replace 尾 -mercaptoethanol, which has similar effect, with less irritating odor and less toxicity. It not only reduced the cost of experiment, but also improved the purity and yield of Illumina sequencing technique to discover the polymorphism of chloroplast genomic DNA. A total of 31 loci were found above the mutation frequency of 2%, of which 199 were located in the gene spacer and 374 were on the gene. And these 374 mutation sites were concentrated on 31 chloroplast genes. The reliability of Illumina sequencing results was verified by Sanger sequencing technology. Sanger sequencing was conducted by screening the appropriate sections with a mutation frequency of 2% -3% and 6% -7%, 20% -23% and 10% -15%, respectively. It was found that the reliability of Illumina sequencing was more than 12%, and many unreliable mutation sites were eliminated, which laid the foundation for further research. The polymorphism analysis of chloroplast gene rps19 and a gene spacer was also provided for the later study. The rps19 gene was only found in 4 mutation sites of 1490 material from different provinces by Sanger sequencing. It may have evolved during a long period of geographic isolation. In the genetic spacer, 52 materials had two identical mutation sites. The result shows that the locus 64298A becomes Tand the locus 64767A becomes C.The other material 887 from Sweden shows more 30 mutation sites. It can be seen that the rate of evolution of the spacer region is faster than that of the functional gene. Regional differences may also be one of the reasons for DNA polymorphism.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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