论文标题:辣椒抗黄瓜花叶病毒（CMV）遗传分析及ISSR分子标记筛选
Resistant Genetic Analysis and Screening ISSR Molecular Marker of Cucumber Mosaic Virus (CMV) in Pepper
论文作者
论文导师 曹碚生；王述彬,论文学位 硕士,论文专业 蔬菜学
论文单位 扬州大学,点击次数 336,论文页数 66页File Size1888K
2007-05-01论文网 http://www.lw23.com/lunwen_2876677/
pepper;;cucumber mosaic virus (CMV);;inheritance analysis;; ISSR molecular marker;;linkage analysis
黄瓜花叶病毒(CMV)是世界上流行最广和研究较多的植物病毒之一,每年在世界范围内对辣椒生产造成严重的危害和损失。目前关于辣椒抗CMV遗传和分子标记筛选的研究报道较少。本文以Vc16a、SS79与SS69构建F2群体,通过对辣椒CMV遗传的研究,揭示其遗传规律,用ISSR方法筛选辣椒与抗CMV基因连锁的分子标记,为辣椒抗CMV育种提供遗传学与分子生物学的理论依据。 1.辣椒抗CMV遗传分析:采用植物数量性状单个世代群体遗传研究方法,对F2世代苗期和成株期发病情况进行分析,研究辣椒抗CMV遗传规律。辣椒苗期和成株期对CMV抗性遗传符合由两对主基因控制的模型,但主基因间表现的作用有所不同。在苗期,05CM-8、05CM-9以及05CM-11的遗传模型都是B-1模型,即主基因间表现为加性-显性-上位性作用;05CM-10符合B-2模型,即主基因间表现为加性-显性作用。05CM-8、05CM-10抗CMV主基因遗传率均为100%;05CM-9和05CM-11抗CMV主基因遗传率也较高,分别为93.97%、99.33%。在成株期也符合由两对主基因控制的模型,但与苗期略有差别:05CM-8与05CM-10的遗传模型都是B-2模型,05CM-9与05CM-11遗传模型都是B-1模型。成株期05CM-8抗CMV主基因遗传率为100%, 05CM-10抗CMV主基因遗传率为95.04%,05CM-9和05CM-11抗CMV主基因遗传率分别为62.91%和61.32%。 2.辣椒ISSR-PCR体系优化:采用4因素3水平[L9(3)4]正交实验设计,由Mg2+、Taq酶、引物和dNTPs组成,对ISSR-PCR体系进行优化,获得最优体系:20μL体系中Mg2+为1.5mmol/L、Taq酶1.5U、引物0.6μmol/L、dNTPs200μmol/L,1×Buffer,50ng模板DNA。 3.辣椒与抗CMV连锁的ISSR分子标记筛选:应用BSA-ISSR技术建立F2代抗感池并筛选抗CMV标记。用40个ISSR引物对Vc16a和SS69及其F2抗感池进行扩增,其中I-3、I-10、I-13和I-34这4个引物在抗感池中扩增出具有多态性条带。这4个引物经F2代单株验证和连锁分析,表明引物I-13和I-34筛选出的标记存在连锁关系,遗传距离为16.3cM,该特异性片段约为1100bp,命名为I-131100;I-34与辣椒抗CMV抗病位点连锁,遗传距离为27.3cM,该特异性片段约为450bp,命名为I-34450。
Cucumber mosaic virus (CMV) is one of the most widespread plant viruses,which has been intensively studied ,caused serious loss in the world.At present, there are few reports about the resistance-inheritance and markers linked to resistant gene of CMV in pepper.This papper analyzes pepper genetic resistance to CMV,and screens ISSR markers linked to resistant gene by using Vc16a、SS79 and SS69.The results will provide theoretical basis about genetics and molecular biology for resistance breeding. 1. Genetic analysis of resistance to CMV in pepper: Disease status in F2 generation was analyzed to reveal genetic resistance at seedling and maturation stages by using segregating generation method of plant quantitative traits.The results indicate that the resistant inheritance controlled by two pairs of major genes in seedling and maturation stages, but there are some differences among the effects of major genes. In seedling stage,the genetic model of 05CM-8,05CM-9and 05CM-11 are all Model B-1, which is additive-dominant-epistatic effects.05CM-10 fits B-2,which is additive-dominant effects. The heritabilities of the major genes of 05CM-8 and 05CM-10 both are 100%, 05CM-9 and 05CM-11 are 93.97% and 99.33%, respectively. In maturation stage,pepper resistance to CMV is also controlled by two pairs of major genes, but there is some differences to that of seedling stage.The genetic models of 05CM-8 and 05CM-10 are model B-2,and 05CM-9 and 05CM-11 are B-1. The major genes’heritabilities are: 05CM-8 100%,05CM-10 95.04%, 05CM-9 62.91%,and 05CM-11 61.32%. 2.Optimization of ISSR reaction system for pepper: In this experiment, orthogonal design with three levels and four factors (Taq DNA polymerase, dNTPs, primer and Mg~(2+)) was used to optimize the pepper ISSR-PCR reaction system. The results showed that a best amplification of ISSR was obtained with the reaction system containing 1×PCR buffer, 1.5mmol/L Mg2+,50ng template DNA of pepper, 1.5U Taq polymerase, 200μmol/L dNTPs, 0.6μmol/L primer in the total volume of 20μL. 3.Screening of ISSR marker: A resistant group and a sensitive group were divided with‘BSA’method to search for the markers with resistance linked to CMV. 40 primers are used to select resistant markers between Vc16a and SS69.Polymorphic products were amplified between the resistant group and the sensitive group by using I-3, I-10, I-13 and I-34 of the 40 primers. Verification and linkage analysis in the F2 generation used the four primers, which indicated that I-13 linked to I-34,and genetic distance was 16.3cM.The polymorphic band was 1100bp and named I-131100;I-34 linked to CMV resistance-site in pepper,and genetic distance was 27.3cM.The polymorphic band was 450bp and named I-34450.
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