ITS序列在三个樱桃栽培种中的遗传多样性分析和应用评价

发布时间：2018-03-21 17:14

本文选题：种质资源　切入点：樱桃　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：中国樱桃(Cerasus pseudocerasus Lindl.)、欧洲甜樱桃(Cerasus avium Linn.)和毛樱桃(Cerasus tomentosa Thunb.)隶属蔷薇科(Rosaceae)李亚科(Prunoideae)樱属(Cerasus),是樱桃的3个重要栽培种。目前对其种间系统关系和种内遗传多样性缺乏基于分子序列的相关深入研究。本研究以中国樱桃、甜樱桃和毛樱桃共95份材料的核糖体内转录间隔区(ITS, internal transcribed spacer)序列作为研究对象,通过分析其序列变异位点,并构建进化树,探讨樱桃种内遗传多样性和它们的系统发育关系,并做二级结构预测验证分析,以揭示中国樱桃、甜樱桃和毛樱桃种内遗传多样性和种间遗传关系,并探讨ITS序列在樱桃遗传多样性及其相关类群的系统进化研究中应用价值。主要的研究结果如下：(1)ITS序列特征。中国樱桃扩增的ITS序列长度范围为702-703bp,甜樱桃的为704-705bp,毛樱桃为709bp,长度变异较小。所有扩增序列对位排列之后长度为712bp,该片段CG核苷酸碱基占总核苷酸碱基的58.1%。共检测到71个变异位点,其中54个属简约信息位点。这些变异位点在ITS1 (37bp-270bp)区中有40个,5.8S (271bp-373bp)区中有3个,ITS2 (374bp-651bp)区有28个。共定义了37个单倍型,其单倍型多样性Hd=0.911,核酸多样性π=0.01444,分离位点多态性平均数θw=0.01954。(2)中国樱桃与欧洲甜樱桃的ITS序列之间的进化距离为0.011-0.027,欧洲甜樱桃与毛樱桃之间的为0.047-0.051,中国樱桃与毛樱桃之间的为0.044-0.057。从ITS序列数据计算的结果来看,中国樱桃与甜樱桃的ITS序列之间同源性较高,毛樱桃则与它们同源性较低。(3)NJ系统发育分析将37个单倍型分为3个分支,Clade I包括中国樱桃的21个单倍型,并获得了93%的自展支持率,Clade II包括了甜樱桃的11个单倍型,自展支持率为96%,Clade III为毛樱桃的5个单倍型构成,自展支持率为100%。(4)在整个单倍型网络中介图中,37个单倍型基因树的拓扑结构呈三臂螺旋分布,毛樱桃的5个单倍型处于臂长较长的一端。网络中介图中共有11处发生冲突,分别位于排列位点79、88、100、130、135、136、153、185、538、641处。在中国樱桃分支中单倍型H2处于中心位置,而且大部分群体中都有分布,是核心的古老单倍型,其余单倍型则分散于该分支的外围,成为网络图的外部节点。(5)二级结构分析。根据进化树中位置和单倍型所代表的样品数量我们选择了H1、H2、H22、H37四个单倍型进行二级结构预测分析,5.8S区域的二级结构基本相同,ITS1区和ITS2区的二级结构差异较大,中国樱桃的单倍型H1、H2最小自由能为-248.00 kcal/mo1和-248.50 kcal/mol,甜樱桃的单倍型H22最小自由能为-251.60kcal/mol,毛樱桃的单倍型H37的最小自由能为-256.5 kcal/mol。将得到的最小自由能进行T检测,三个物种的单倍型之间的最小自由能在P0.05时差异显著。它们的下半部分的茎环结构较为相似,上半部分则有较大差异,结合最小自由能数据和结构图,支持基于ITS序列所做的进化树。
[Abstract]:Cerasus pseudocerasus Lindl.China (Cerasus avium Linn.) and Cerasus tomentosa Thunb.) belong to the genus Prunoideae (Rosaceae), and are three important cultivated species of Cherry. At present, the interspecific phylogenetic relationship and intraspecific genetic diversity are not based on subdivision. Further study on the correlation of subsequences. The internal transcribed spacer (internal transcribed spacer) sequences of 95 samples of sweet cherry and hairy cherry were studied. The genetic diversity and phylogenetic relationship among them were studied by analyzing the sequence variation sites and constructing an evolutionary tree. In order to reveal the intraspecific genetic diversity and interspecific genetic relationship of Chinese cherry sweet cherry and hairy cherry the secondary structure prediction and verification analysis was done to reveal the genetic diversity of Chinese cherry sweet cherry and hairy cherry. The application value of ITS sequence in the phylogenetic study of cherry genetic diversity and its related groups was discussed. The main results were as follows: the ITS sequence length of Chinese cherry was 702-703bpand that of sweet cherry was 704-705bp. The length of Cherry was 709 BP, the length of which was 712 BP after all the amplified sequences were arranged in opposite position. The CG nucleotide base of this fragment was 58.1% of the total nucleotide base. A total of 71 mutation sites were detected. Among them, 54 belong to parsimonious information loci. These variation loci are 28 out of 40 in the ITS1 37bp-270bp) region, and there are 3 ITS2,374bp-651bp) regions. 37 haplotypes have been defined. Its haplotype diversity is 0.911, nucleic acid diversity 蟺 0.01444, and the average polymorphism of segregation sites 胃 w 0.01954.02) the evolutionary distance between Chinese cherry and European sweet cherry ITS sequence is 0.011-0.027, between European sweet cherry and hairy cherry is 0.047-0.051, between Chinese cherry and hairy cherry is 0.047-0.051, between Chinese cherry and European sweet cherry is 0.047-0.051, between European sweet cherry and European sweet cherry is 0.047-0.051. Between 0.044-0.057. from the results of the ITS sequence data calculations, The ITS sequence of Chinese cherry and sweet cherry was highly homologous, while that of hairy cherry was low. NJ phylogeny analysis showed that 37 haplotypes were divided into 3 branches, including 21 haplotypes of Chinese cherry. The self-development support rate of 93% was 93%, which included 11 haplotypes of sweet cherries, and the self-exhibition approval rating of 96 Clade III was 5 haplotypes of hairy cherries. In the whole haplotype network intermediary map, 37 haplotype gene trees have a three-arm spiral distribution, and five haplotypes of hairy cherry are at one end of longer arm length. There are 11 conflicts in the network intermediary diagram. The haplotype H2 is located in the central position of the Chinese cherry branch, and is distributed in the majority of the population, which is the core of the ancient haplotype, while the other haplotypes are scattered in the periphery of the branch. Based on the location of the evolutionary tree and the number of samples represented by the haplotype, we selected four haplotypes H1H2H2H22H37 to predict the secondary structure and analyze the basic phase of the secondary structure in the 5.8S region. The secondary structure of ITS1 region and ITS2 region were different greatly. The minimum free energy of haplotype H _ 1O _ 2 was -248.00 kcal/mo1 and -248.50 kcal / mol, that of sweet cherry was -251.60 kcal / mol, and that of haplotype H37 was -256.5 kcal / mol. The minimum free energy (MEF) of the haplotypes of the three species was significantly different when P05. The structure of the stem ring in the lower part of the species was similar to that in the upper part, which combined with the minimum free energy data and the structure diagram. Support for evolutionary trees based on ITS sequences.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S662.5

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