【作者】 汪志伟；

【导师】 向长萍；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2006， 博士

【摘要】 萝卜（Raphanus sativus L．）是深受世界人民喜爱的根菜类蔬菜。萝卜的杂种优势非常显著，能够有效地提高产量和改善纤维品质。在植物中，细胞质雄性不育（Cytoplasmic male sterility，CMS）是一种不能产生正常功能花粉的母性遗传性状，被广泛地应用于商业化生产F₁代杂种。萝卜CMS系的细胞质即能导致萝卜属植物雄性不育，又能导致芸薹属植物雄性不育，而且其CMS不易受环境条件和不同遗传背景的影响，从而是十字花科植物中最受关注的不育细胞质之一。目前已有一种萝卜CMS及其对应的恢复位点从萝卜导入到油菜中，但一些不良的农艺性状随着恢复基因的导入而出现。远期目标在于阐明萝卜CMS及育性恢复以及使萝卜CMS在十字花科杂种优势育种中得到充分利用。本篇论文中，我们就萝卜CMS胞质不育和核基因的分子标记及其分子特征进行了描述。取得的主要研究结果如下： 获得了一个萝卜CMS系统，该系统由雄性不育系9802A、保持系9802B和恢复系9802H组成。该不育系花药外观皱缩或正常，无可见花粉，而Ogu CMS系花药外观皱缩未完全发育；NWB CMS系则花药外观正常、花药内有部分花粉。不育系9802A和恢复系9802H杂交产生的F₁植株全部表现雄性可育，表明CMS的恢复表现型是一种显性性状。获得了由300个单株组成的F₂代育性分离群体。卡平方测验表明分离群体中可育与不育的分离比符合3∶1的比例，证明9802A雄性不育的育性恢复受单一显性基因控制。 用萝卜Ogu orf138基因的特异引物组合ORF-F和ORF-R，在萝卜CMS系9802A总DNA中扩增出了一条DNA片段，而在保持系9802B和恢复系9802H中未扩增出任何产物。序列分析表明该片段与特异诱导Ogu CMS相关基因orf138序列完全一致。该DNA片段可以作为选育萝卜CMS的分子标记。 合成了能够扩增出萝卜NWB CMS系的特异分子标记的引物组合NWB-F和NWB-R，该引物组合在萝卜CMS保持系9802B和恢复系9802H总DNA中扩增出了单一DNA片段，而在CMS系9802A总DNA中未扩增出任何产物。该DNA片段可以作为辅助选育萝卜CMS保持系的分子标记。 采用混合分群分析法和F₂代的300个单株筛选了萝卜CMS恢复基因Rfw的RAPD标记。对100条RAPD引物的扩增产物进行筛选后，发现RAPD标记OPC06-1900与恢复基因Rfw连锁。通过分子克隆和核苷酸测序将该RAPD标记转换成了显性的SCAR标记SCC06-1894。BLAST搜索表明SCC06-1894与拟南芥和甘蓝硫酸盐转运蛋白基因的对应区域高度同源。设计了等位特异引物，扩增出了SCAR标记SCC06-415。用F₂代的分离群体进行PCR分析及序列分析表明SCC06-1894和SCC06-415等位，与Rfw／rfw的遗传距离为8.0cM。根据一个萝卜更多还原

【Abstract】 Radish （Raphanus sativus L.） is a popular root vegetable throughout the world. The heterosis in radish is much significant, especially in increasing yield and improving fiber quality. In plants, cytoplasmic male sterility （CMS） is a maternally inherited trait which is characterized by the inability to produce functional pollen and CMS systems are widely used for the production of commercial F₁ hybrid plants. In Cruciferous plants, radish male sterile cytoplasm is one of the most intensively investigated cytoplasms, because the cytoplasm causes male sterility in not only Raphanus but also Brassica species, and the expression of CMS was not affected by different environmental conditions and different genetic backgrounds. A radish CMS and the corresponding nuclear restorer locus have been introgressed from radish into rapeseed （B. napus L.）, but deleterious agronomic characters were also introduced along with the Rf （fertility restorer） gene. Long term goals are to clarify the molecular mechanism of male sterility and fertility restoration in radish and to take full advantage of the radish CMS to breed F₁ hybrid plants in Cruciferous plants. In this thesis, development and characterization of molecular markers of cytoplasmic sterile and nuclear genes for CMS in radish were described. The main results obtained are listed below:A radish CMS system was gained and consists of male sterile line 9802A, maintainer line 9802B and restorer line 9802H. F₁ plants from cross between male sterile line 9802A and restorer line 9802H were all male fertile, indicating that the male fertile type is a dominant character. A F₂ population composed of 300 individuals was constructed. The segregating population was found to fit a segregation ratio 3:1 for either sexual type （male fertile : male sterile）. x² test value was highly significant in the F₂ population （x²=0.22, 0.70>p>0.50）. These results confirmed that male fertility is recovered by a single dominant gene.Using PCR, we determined that the radish Ogu orf138 gene specific primer pair ORF-F/R resulted in the amplification of a DNA fragment in the radish CMS line 9802A, but no PCR products were detected in the maintainer line 9802B and in the restorer line 9802H. Sequence analysis demonstrated that the DNA fragment was identical to the Ogu orf138, indicating the presence of the orf138 gene, the specific factor inducing Ogu CMS, in the CMS line 9802A. This DNA fragment can be used as a DNA marker to select CMS line in a radish breeding program.Primer pair amplifying a molecular marker specific to the NWB CMS line in radish更多还原