【作者】 李培；

【导师】 龚义勤；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2006， 硕士

【摘要】 萝卜（Raphanus sativus L．）是一种非常重要的蔬菜作物，其具有明显的杂种优势，以细胞质雄性不育系为母本，实行三系配套的制种方式是萝卜杂种优势利用的重要途径。本研究利用多个雄性不育系与保持系配制的子代群体，筛选恢复基因，将其与不育系进行杂交，对杂种和不育系母本进行差异显示；建立适用于萝卜的RAMP体系，并应用于萝卜的可育与不育品种；综合利用光镜、电镜和差异显示技术对中国萝卜的CMS性状进行研究。 提取萝卜嫩叶总DNA，利用针对orfl38基因区域设计的两套引物对供试品种进行检测，得到长度约为780bp和280bp的扩增产物，这两种产物的扩增结果在材料中表现一致。将保持系B₁与不育系A₁，A₅中的780bp扩增产物进行回收、纯化和测序，发现分别与报道的orfl38基因序列同源性为100％，且不同材料中的orfl38基因差异不大，同源性为97.75％。同时利用针对恢复基因的连锁STS标记设计的引物，在供试材料中对恢复基因进行筛选，结果发现只有“东方黑大根”具有恢复基因。雄性不育受环境的影响，不育系中会出现可育植株，而保持系中也会出现不育植株，将育性变异的植株进行orfl38基因和恢复基因的筛选，结果与育性正常的植株一致，可能是只与花粉有关的一些因素发生了变化。将“东方黑大根”与不育系进行杂交形成杂交种，对杂交种和母本不育系进行差异显示比较，结果发现差异条带多为不育系具有，经过比较分析，差异条带多植物中的核糖体RNA同源性较高。 建立适合于萝卜的分子标记RAMP体系。对RAMP分析体系中的Mg²⁺、dNTPs和引物浓度进行优化，分别设计3个浓度梯度：mg²⁺为0.75，1.5，3.0mmol·L^-1；dNTPs为0.05，0.15，0.3mmol·L^-1；引物为0.065，0.2，0.4μmol·L^-1，并对合适的复性温度进行研究。筛选出的RAMP优化体系为（20μl）：dNTPs 0.15mmol·L^-1，Mg²⁺1.5mmol·L^-1，引物0.2～0.4μmol·L^-1，DNA 10ng，Taq E 0.8U；PCR扩增程序为94℃3min，94℃ 1min，45℃ 1min，72℃ 1.5min，42个循环，72℃ 8min。运用此体系，进行引物组合筛选，并对7个萝卜品种的遗传多样性与品种鉴定进行RAMP标记分析。将建立的RAMP体系应用于萝卜的雄性不育，发现不育系与保持系之间没有明显的差异，而具有恢复基因的“东方黑大根”与其余没有恢复基因的品种之间有明显的差异，说明RAMP标记可以应用于萝卜恢复基因的确定。更多还原

【Abstract】 Radish （Raphanus sativus L.） is one of the most important vegetable crops, it shows remarkable heterosis, producing hybrid seed using Cytoplasmic Male Sterile（CMS） line as female parent is a very important way to utilize the heterosis in radish. In this study, screening the Restore fertility（Rf） gene, hybrid the CMS line and the variety with Rf, using the DDRT-PCR in the hybrid and female parent; constructing the RAMP system in radish, using it in radish CMS; light-microscopy, electro-microscopy and DDRT-PCR were performed to detect the mechanism of CMS in radish maked by many CMS line and maintainer line.The total DNA were isolated from young radish leaves, two DNA fragments about 780bp and 280bp were amplificated in primer to orf138 gene region, the two fragments exit or not simultaneity in radish. After recovering the 780bp fragment in maintainer line B₁ and CMS line A₁ and A₅, blast result shows that the three fragments is 100% homology with the reported orf138 nucleotide sequence, separately. There is no difference among the three fragments, they are 97.75% homology each other. Screening the Rf gene though the primer to STS marker linked to Rf gene, the Rf gene about fragment 190bp was discovered in "Dong fang hei da gen" only. The sterility was influenced by environment, the fertility may be changed in CMS line and maintainer line. The fragments about 780bp, 280bp and 190bp exiting or not in the radish which fertility change is same as those fertility is common. Maybe the factor related to pollen has changed. Hybrid the CMS line and "Dong fang hei da gen", after comparing the hybrid and female parent though DDRT-PCR, the difference bands exit in female parent mostly, the homology is high between the RNA in plant.The Random Amplified Microsatellite Polymorphism （RAMP） system suitable to radish was constructed. Genome DNA of Radish was analyzed by optimizing the concentration of Mg²⁺, dNTP and primer in. Three different concentrations level were set respectively, Mg²⁺ 0.75, 1.5, 3.0mmol · L^-1; dNTP 0.05, 0.15, 0.3 mmol· L^-1; primer 0.065, 0.2, 0.4 μmol· L^-1, and the suitable annealing temperature was also screened. The更多还原