【作者】 尹昭坤；

【导师】 袁潜华； 廖承红；

【作者基本信息】 海南大学 ， 作物遗传育种， 2012， 硕士

【摘要】 本文以海南普通野生稻和山栏稻为实验材料,以核糖体转录间隔区(ITS,internal transcribed spacer)序列为研究对象,构建海南普通野生稻和山栏稻的分子系统进化树,找寻区分籼粳业种的特异位点,研究普通野生稻不同居群或品种间的亲缘关系。以促进对海南普通野生稻和山栏稻遗传背景的了解,探讨稻属亚种间的系统进化研究手段和方法,为普通野生稻和山栏稻资源评价与利用提供分子遗传学指标。主要研究结果如下：(1)ITS序列变异统计。克隆分析了来自海南普通野生稻和山栏稻30份材料的ITS区和5.8S区序列。5.8S rDNA的长度范围在86bp-169bp。G/C含量相差不显著。海南普通野生稻和山栏稻ITS1区长度范围在163bp-239bp,G/C含量差异达到40%。ITS2区长度范围在165bp-243bp,G/C含量差异达到31.8%。从ITS1和ITS2片段长度多态性来看,ITS2区的变异要大于ITS1区。(2)ITS多态性分析。海南普通野生稻和山栏稻的rDNA5.8S基因在编码区共有77个信息位点,核酸多态性(Pi)为0.42671,ITS区共有281个信息位点,核酸多态性(Pi)为0.63270。rDNA5.8基因和ITS区在海南普通野生稻居群内部和山栏稻品种内部的变异要大于它们各自与栽培稻间的变异。基于ITS序列构建的Maximum-Likel-ihood系统树中,30个野生稻居群、山栏稻和栽培稻品种按籼粳类群分开,而并非按照地理来源或稻作区聚类。其中有7份海南普通野生稻居群和1份山栏稻品种偏籼,有19份普通野生稻和2份山栏稻品种偏粳。普通野生稻、山栏稻籼粳差异要大于地区间差异,说明海南普通野生稻各居群间和山栏稻各品种间已发生籼粳分化。(3)关于籼粳特异位点。研究发现在整个海南普通野生稻居群和山I栏稻品种间,ITS区域的信息位点丰富,居群间差异较大。海南普通野生稻ITS1和ITS2区内信息位点的百分率分别达到30.7%和39.6%。山栏稻品种间信息位点分别达到了29.4%和32.6%。本研究还发现了17个判定籼粳表征性的特异位点,有效验证了应用ITS序列进行属内种间分类的可靠性。(4)山栏稻与海南普通野生稻的进化关系。综合分析山栏稻与海南普通野生稻ITS区碱基突变分布和ITS1、ITS2序列RNA二级结构,海南普通野生稻较山栏稻品种具有更为丰富的突变类型,且包含了山栏稻品种的所有变异。RNA二级结构分析中,ITS2较ITS1区具有更强的保守性,并发现了山栏稻品种的特异序列和2个ITS2区保守的特异基序,推测山栏稻与海南普通野生稻发生过基因交流,存在基因渗入现象,且野生稻处于遗传进化的中心。更多还原

【Abstract】 In this study, we sequenced Internal Transcribed Spacer (ITS) of different population of Hainan common wild rice(Oraza rufipogon) and Shanlan upland rice (Oraza sativa). By DNA sequence analysis, we attempted to construct the phylogenetic tree and establish a molecular marker to distinguish Indica and Japonica rice subspecies, so as to constitute a new research method of the oryza genus and promote the understanding of the genetic background of the primary Oraza rufipogon and shanlan upland rice and offer a molecular genetic marker for the evaluation and utilization of rice genetic resources. Main results are showed as follows:1. Sequences of the entire ITS region, including ITS1, ITS2, from the experimented materials was amplified, cloned and sequenced. The range of length of5.8S was86bp to169bp. There was no significant different of GC content in this fragment. The sequences of ITS1were from163bp to239bp. The GC contents were44.1%-77.2%. The sequences of ITS2ranged from165bp to243bp. In size and G/C content varied from44%to71%. Mutations sites of ITS2were significantly higher than that of ITS1.2. ITS sequences of the experimented materials were reconstructed by the Molecular Evolutionary Genetics Analysis version5(MEGA5) for building sequence alignments and phylogenetic trees. Of the aligned positions, there were358informative sites (124in ITS1,157in ITS2and77in5.8S). Nucleotide diversity (Pi) was0.42671in5.8S and0.6327in ITS of rDNA. It had indicated that the population genetic diversity level of the ITS region in Oraza rufipogon were higher than those between Oraza rufipogon and cultivated rice. Maximum-Likelihood phylogenetic tree based on ITS sequenceing revealed that30species of wild rice and shanlan rice varieties was clustered together by indica-japonica differentiation. The grouping of the lines was not in accordance with the available pedigree date on their geographic origin and rice regions. Phylogenetic analyses also revealed those seven common wild rice populations and one shanlan upland rice variety had a closed phylogenetic relationship with indica rice. Nineteen matericals and two materials of shanlan upland rice had a closed phylogenetic relationship with japonica rice.3. In this study, the informative sites of ITS1and ITS2account for30.7%and39.6%of the total length respectively in common wild rice, and account for29.4%and32.6%in shanlan upland rice. Seventeen subspecies differentiating sites were screened betteen common wild rice and shanlan upland rice. The feasibility of the investigation of relationships between species in the genus was confirmed by using highly and moderately repetitive DNA sequences, rDNA ITS.4. Comprehensive analysis of mutation distribution and secondary structure of RNA of ITS region between shanlan rice and the common wild rice. The results showed that constrasting with shanlan upland rice, there was a great diversity of ITS region among the common wild rice. Four ITS variation types were found in common wild rice, including two variation types in shanlan upland rice. Analyses of secondary structure of RNA of ITS showed that ITS2sequence is very conservative in the secondary structure of RNA. Specific variation types from shanlan upland rice and two conservative motifs were fond in ITS2regions. It could speculate out that they had happened gene exchange and introgression, and common wild rice appeared to be the center of genetic diversity.更多还原