【作者】 姚明华；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2013， 博士

【摘要】 黄瓜花叶病毒(Cucumber mosaic virus, CMV)是辣椒病毒病的主要毒源之一既影响辣椒的产量又影响辣椒的品质,主要表现为使辣椒果实畸形,果实商品性丧失,严重时甚至导致绝产。因此,辣椒抗CMV育种已成为辣椒抗病育种的主要目标之一开展辣椒抗CMV育种前,必须首先明确当地病毒分离物亚组分化情况,才能有针对性的开展辣椒抗CMV育种工作。应用RT-PCR技术能够快速、准确的检测植株体内CMV分离物亚组归属。利用分子标记技术构建辣椒遗传连锁图谱,并利用QTL分析软件鉴定抗病相关位点,能够大大提高育种中对数量性状基因型选择的准确性和预见性,加速育种进程。本研究对湖北地区侵染辣椒CMV分离物亚组进行鉴定；筛选辣椒抗CMV苗期接种鉴定方法,并进行抗(耐)CMV辣椒材料的鉴定；以抗CMV辣椒材料P0747和感CMV的材料P0630及其杂交的后代分离群体为材料,对其抗性遗传规律进行研究,并应用SSR和ISSR两种分子标记技术构建了辣椒分子遗传连锁图谱,对CMV抗性基因进行了QTL定位分析,对辣椒抗CMV育种具有重要的指导意义。主要研究结果如下：(1)根据已报道的CMV外壳蛋白(CP)基因序列设计引物,应用RT-PCR方法,对湖北地区侵染辣椒的CMV分离物的病毒外壳蛋白基因进行基因扩增和序列分析。结果表明,湖北省内不同辣椒主产区采集到的样品CP基因之间的核苷酸同源率高达98.2%-99.2%,分离物均属于亚组IB成员。(2)筛选出辣椒抗CMV苗期人工接种鉴定最佳条件为：在辣椒苗龄为4片真叶,采用摩擦接种法、接种温度30℃环境条件下,可以达到最佳的鉴定结果；应用苗期人工接种鉴定方法从供试的74份辣椒种质资源中筛选出抗CMV材料2份,耐CMV材料26份。(3)利用6份对CMV抗感程度不同的辣椒材料通过Griffing的完全双列杂交法对辣椒抗CMV的遗传规律进行了研究,明确了辣椒对CMV优势育种中亲本的选择和选配原则；再利用采用植物数量性状主基因+多基因混合遗传的世代联合分析方法,对辣椒抗CMV材料的抗性遗传规律进行分析,明确了辣椒对CMV的抗性受“两对加性-显性-上位性主基因+加性-显性多基因”控制,并对各遗传模型中抗性表现的主基因遗传率和多基因遗传率以及基因间的互作效应进行了详细解析。(4)应用SSR和ISSR两种分子标记方法构建了辣椒遗传连锁图谱,该图谱包括17个连锁群,共有140个标记进入连锁群,总长度为1420.5cM,标记间平均图距为10.1cM。(5)应用构建的辣椒分子遗传连锁图谱,结合复合区间作图法对辣椒抗CMV-HB相关QTL进行分析,共定位了6个QTL,分别为qcmv.hb-4.1、qcmv.hb-7.1、 qcmv. hb-8.1、qcmv.hb-8.2、qcmv.hb-8.3和qcmv.hb-16.1,位于4个连锁群上,能够解释表型变异的2.8～43.5%,其中qcmv.hb-4.1和qcmv.hb-8.2在夏季和秋季接种试验中均能够被检测到,其中qcmv.hb-8.2能够解释37.7～43.5%的表型变异,可以认为是一个控制辣椒对CMV-HB株系抗性的主效QTL,加性效应为正,加性效应值为0.59～0.64,当qcmv.hb-8.2表现抗病亲本基因型时,能够降低病情指数。qcmv.hb-4.1能够解释10.7-11.2%的表型变异,加性效应为正,加性效应值为0.24～0.25,当qcmv.hb-4.1表现抗病亲本基因型时,同样能够降低病情指数。更多还原

【Abstract】 Cucumber mosaic virus (Cucumber mosaic virus, CMV) is the main virus type leading to the virus disease on pepper. This disease affects both yield and quality in pepper production. The symptoms in field mailnly show malformation of fruits, loss of commodity or even never produced. In order to overcome the damage of CMV disease, breeding cultivars with CMV resistance is becoming one of main targets in pepper breeding.For CMV-resistance breeding on pepper, we should understand the subgroup of CMV infecting pepper plant. The technology of the real-time quantitative PCR can be used to identify the subgroup of CMV in plant more rapidly and accurately. The utilization of molecular markers can be used to build genetic maps of pepper, and the quantitative trait loci relative to CMV-resistance also can be scanned by QTL analysis softwear. With the help of the molecular markers linked to CMV-resistance QTL, the accuracy and foreseeability for breeding selection on genetype of quantitative trait will be improved, and the efficiency for breeding on pepper is also increased.In this study, the subgroup and strain of CMV infecting pepper plant in Hubei province was identified firstly. Secondly, we screened the optimal condition of seedlings inoculation identification for pepper CMV disease and we also evaluated the CMV resistance of germplasm resources on pepper. Thirdly, two inbred lines of the pepper varieties, a CMV-resistance homozygous P0747and a CMV-susceptible line P0630, and their segregation populations were developed. Both parents and their segregation populations were used for inheritance analysis and genetic map construction. Finally, the QTLs relation to CMV-resistance was located by CIM method. The results of QTLs analysis will guide the CMV-resistance breeding on pepper.(1) Using the RT-PCR method and primers designed from the sequence of CP genes, the CMV isolates in Hubei area was identified. The results were as follows:the CP genes of CMV isolates in Hubei area shared98.2%-99.2%homology with those of CMV subgroup IB strains from Genbank. It was determined that all of the CMV isolates belonged to the subgroup IB of CMV.(2) The optimal condition of seedlings inoculation identification for pepper CMV disease is as following:The youth test peppers at4leaf-stages were dusted with600-mesh carborundum and inoculated by rubbing the leaves with inoculums. After inoculation, pepper seedlings were kept in a plastic house maintained at25℃to30℃. Totally seventy-four inbred lines were identified for CMV-resistance, and2inbred lines were resistant to CMV,26inbred lines moderate resistant.(3) Genetic analysis on the resistance of CMV by Griffing’s complete diallel crosses. The results of the analysis cleared the principle of parent choice in cross breeding of disease-resistance. The two parental lines and their segregation populations obtained from a cross between them were used for inheritance analysis by the joint segregation analysis method. Our results revealed that CMV resistance in’P0747’is controlled by two partially additive-dominant major genes and additive-dominance polygenes and the polygenes effect existed at the same time.(4) A F2population was used for constructing linkage groups by SSR and ISSR methods. This map included140markers mapping onto17LGs, covering1420.5cM according to the Kosambi function. The average distance between the markers was10.1cM.(5) QTLs were detected by the composite interval mapping (CIM) method based on the linkage groups. A total of six QTLs, qcmv.hb-4.1, qcmv.hb-7.1, qcmv.hb-8.1, qcmv.hb-8.2, qcmv.hb-8.3and qcmv.hb-16.1, were identified on4LGs (LG4, LG7, LG8and LG16) in two inoculation experiments in2009, which accounted for2.8-43.5%of the individual phenotypic variations. Notably, the QTL qcmv.hb-8.2was repeatedly identified in two seasons and showed the largest effect and explained37.7-43.5%of the total phenotypic variation, with the allele from’P0747’increasing CMV resistance by0.59-0.64. The QTL qcmv.hb-4.1also had a sizable effect and could explain11.2%of the variation in summer and10.7%of the variation in autumn, with the allele from’P0747’ increasing CMV resistance by0.24-0.25.更多还原