【作者】 李红双；

【导师】 李锡香；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2009， 博士

【摘要】 萝卜（Raphanus Sativus L.）为十字花科萝卜属蔬菜作物,栽培历史悠久,在我国蔬菜生产中一直占有重要地位。萝卜病害一直是影响我国萝卜生产的重要因素,其中,发生最为普遍同时危害也最为严重的是芜菁花叶病毒（TuMV）病和黑腐病两大病害。为了实现对病害的有效防治,提高萝卜产品的品质和产量,最根本的解决办法是培育抗病品种。我国作为萝卜的起源地之一,种质资源丰富。开展萝卜资源对TuMV和黑腐病的抗性鉴定和评价,筛选优异抗源,解析其抗性遗传规律和挖掘抗性基因,对萝卜抗病基础理论研究和抗病遗传育种均具有重要的理论和现实意义。基于以上背景和目的,本研究对前人初步鉴定评价获得的部分代表性的萝卜抗、感种质资源进行TuMV和黑腐病抗性的重复鉴定,筛选出典型抗、感种质资源;在此基础上进行萝卜特定抗源对TuMV和黑腐病抗性遗传规律研究;通过分子遗传图谱的构建、QTL定位分析以及分子标记的方法,对萝卜TuMV和黑腐病抗性基因的遗传进行解析,挖掘抗病基因源。主要研究结果如下:1.萝卜代表性种质对TuMV和黑腐病抗性的重复鉴定:在萝卜种质对TuMV和黑腐病田间和苗期抗性初步鉴定筛选的基础上,对其中的26份抗、感特性存在明显差异的自交系进行了苗期抗性重复鉴定。筛选出对TuMV高抗的材料12份,抗病材料8份,中抗材料5份,感病材料1份;对黑腐病抗病的材料3份,中抗材料6份,感病材料6份,高感材料11份。在这些材料中,发现对TuMV和黑腐病均表现抗病的材料2份,均表现感病的材料1份。2.萝卜对TuMV和黑腐病抗性的遗传规律研究:采用完全双列杂交的配合力分析法对萝卜抗TuMV和黑腐病的遗传规律进行了初步研究,明确了在萝卜对TuMV和黑腐病抗病优势育种中亲本的选择和选配原则。同时,利用数量性状的主基因+多基因混合遗传的世代联合分析法,对萝卜抗两种病害的遗传特性进行了进一步解析,明确了萝卜对TuMV的抗性遗传符合“两对加性-显性-上位性主基因+加性-显性-上位性多基因”遗传模型;对黑腐病的抗性遗传符合“一对加性-显性主基因”遗传模型,同时存在多基因效应。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。各世代抗病毒病主基因遗传率在55 %～95 %之间,多基因遗传率为0～40.9 %,环境方差仅占总方差的4.3 %～10.1 %;F2世代抗黑腐病主基因遗传率为72.4%,多基因遗传率为9.07%,环境方差占表型方差的比率为18.5%。3.萝卜分子遗传图谱的构建:利用同时对萝卜TuMV和黑腐病存在明显抗性差异的自交系构建了包括360个单株的F2分离群体,以此群体为研究对象,首次采用优化的萝卜SRAP分子标记技术和SSR分子标记技术相结合的方法,构建萝卜分子遗传图谱,该图谱包括9个连锁群,由196个标记组成,图谱总长度736.2 cM,平均图距3.76 cM。4.萝卜对TuMV和黑腐病抗性基因的QTL定位和分子标记:应用构建的萝卜分子遗传图谱,通过多QTL模型作图法,首次对控制萝卜TuMV和黑腐病的抗性基因进行了QTL定位与遗传效应分析,共发现了控制萝卜对TuMV抗性和黑腐病抗性的4个QTL,这4个QTL分布在LG3和LG5连锁群上。在控制萝卜对TuMV抗性的2个QTL中,1个为增效位点,1个为减效位点,QTL的贡献率分别为7.3 %和11.7 %;控制黑腐病抗性的2个QTL中,有1个为增效位点,QTL的贡献率为26.6%,1个为减效位点,QTL的贡献为45.3 %。同时采用混合分组分析法（BSA）法对萝卜TuMV不同抗源的抗性基因进行分子标记研究,结果找到一个与抗病基因连锁的分子标记CoMe7F/BEm12R-120,连锁遗传距离为7.9 cM。5.与无毒基因对应的萝卜抗黑腐病基因的分子标记:首次应用从黑腐病菌Xcc₈004中分离出来的8个含有特定无毒基因的菌株对20份萝卜黑腐病抗、感材料进行筛选,发现有12份材料对不同的无毒基因菌株存在抗性反应,表明这12份材料中含有与相应的无毒基因对应的抗性基因。同时,应用以KB07-3和KB07-10为亲本构建的F2分离群体,采用BSA方法和SRAP分子标记技术对与无毒基因Xcc₃176对应的抗病基因进行了分子标记研究,找到了一个与该抗病基更多还原

【Abstract】 Radish （Raphanus sativus L.） is a kind of cruciferous vegetable crops, and it has a very long history of cultivation and has occupied an important position in the vegetable production of China. Disease in radish has been always an important factor affecting the production of radish in China, and the most prevalent and serious diseases are turnip mosaic virus （TuMV） and black rot. In order to achieve effective prevention and treatment of the diseases and improve the quality and yield of radish, the most fundamental solution is to develop resistant varieties. China is one of the origins of radish and it is rich in radish genetic resources, so it has great significance to evaluate the resistance of the rich germplasm resources to TuMV and black rot, discover elite resistant genetic resources, further clarify its resistance inheritance and mine resistant genes for disease-resistance theory study and radish breeding practice.Based on above background and purpose, a repeated identification was carried out on the resistance of representative radish germplasm to TuMV and black rot, which were preliminarily identified in the previous study in our lab.Based on the typical resistant and susceptible radish germplasm screened out, the resistance inheritance of the given resistant genetic resources to TuMV and black rot were studied at the phenotype, and their resistant inheritance were further analyzed at molecular level by constructing radish molecular genetic map, locating QTLs and developing molecular markers. The main conclusions of this paper are as follows:1. Repetitive identification on the resistance of representative radish gerplasm to TuMV and black rot: The twenty-six inbred lines with obvious difference in the resistance to TuMV and black rot were repeatedly indentified in the light of previous preliminary identification in field conditions and at seedling stage.The result showed that 12 inbred lines were high resistant to TuMV , 8 inbred lines resistant, 5 inbred lines moderate resistant and 1 inbred lines susceptible; and 3 inbred lines were resistant to black rot , 6 inbred lines moderate resistant , 6 inbred lines susceptible, 11 inbred lines high susceptible. From the results, we found 2 inbred lines were resistant both to TuMV and black rot, and 1 sample was susceptible both to TuMV and black rot among them.2. Genetic analysis on the resistance of the given resistant germplasm to TuMV and black rot at the phenotype level: The resistance inheritance of radish germplasm to TuMV and black rot were studied through the combining ability analysis of complete diallel cross. It was made definite that the resistances to both diseases were dominant or partly dominant to susceptibility. The principle of parent choice and matching on the basis of the S.C.A and G.C.A was stressed in cross breeding of disease-resistance. The resistance inheritance of typcal resistant resources to TuMV and black rot was further analyzed using the joint segregation analysis of a mixed genetic model of major gene plus polygene in generations. It was proved that the inheritance of radish resistance to TuMV obeyed the“two pairs of additive - dominance– epistasis major gene plus additive - dominance– epistasis polygenes”genetic model; and the resistance inheritance of radish to black rot disease obeyed the“a pair of additive - dominance major gene”genetic model, and the polygenes effect existed at the same time. and the major gene heritability（MGH）, the polygene heritability（PGH） and the ratio of environmental variance （EV） to total variance （TV） were estimated on the base of the genetic models. The MGH and the PGH for the resistance to TuMV were respectively 55 %⁹5 % and 0⁴0.9 % , the ratio of EV to TV was 4.3 %¹0.1 % in different segregation populations; MGH and PGH for the resistance to black rot were 72.4% and 9.07%, the ratio of EV to TV was 18.5% in F2 population.3. Construction of radish molecular linkage map: A F2 segregation population was established applying two radish inbred lines which were obviously different in the resistance both to TuMV and black rot. A molecular genetic linkage map of radish was constructed on the basis of the population by two optimized molecular marker methods of SRAP and SSR. The molecular genetic linkage map included nine linkage groups and 196 markers, its total length was 736.2 cM, with an average map distance of 3.76 cM.4. QTL Mapping and molecular tagging of resistant genes to TuMV and black rot: The QTLs and their genetic effects of the genes resistant to TuMV and Black rot were mapped and dissected on the basis of the radish molecular genetic linkage map and with the multi-QTL model. It was found that 4 target QTLs intensively locating in LG3 and LG5 linkage groups. Among them 2 QTLs were related to the resistance to TuMV, of which one was effect-enhancing with the contribution rate of 7.3 %, another was the negative-effect with the contribution rate of 11.7 %. And 2 QTLs were related to the resistance to black rot, of which one was effect-enhancing QTLs and one was the effect-reducing QTL,the contribution rate was 26.6 % and 45.3 %.Meanwhile, the resistance gene to TuMV was tagged with help of the bulk segregation analysis （BSA）. It was found that a molecular marker CoMe7F/BEm12R-120 was linked to resistance gene with the genetic linkage distance is 7.9 cM.5. Molecular tagging of resistant genes corresponding to avirulence genes of black rot pathogen in radish:Twenty radish inbred lines with obvious difference in resistance to black rot were screened with 8 strains containing different avirulence genes that were isolated from black rot pathogen Xcc₈004. It was demonstrated that 12 lines of them appeared resistant response to different avirulence gene strains, suggesting the existence of corresponding resistant genes. The molecular marker for the resistant gene corresponding to avirulence gene Xcc₃176 was studied in a segregation population F2 constructed from KB07-3 and KB07-10. By using of the BSA method and SRAP molecular marker system, a molecular marker CuMe6F/CoEm11R-260 was obtained, which was closely linked to the resistant gene with a genetic linkage distance of 7.6 cM.更多还原