【作者】 朱献文；

【导师】 龚义勤； 柳李旺；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2006， 硕士

【摘要】 萝卜品种的改良依赖于品种资源的收集、整理、分析与创新。综合应用多种分子标记对萝卜种质资源遗传多样性进行分析、并对品种进行鉴定，通过萝卜花药培养单倍体诱导技术与秋水仙素加倍技术从倍性水平上对萝卜品种进行操作，对萝卜品种改良具有重要意义。本研究利用107个RAPD随机引物、4组AFLP引物与13对SRAP引物对17个国内外具有代表性的萝卜品种进行遗传多样性与种质鉴定研究，结果显示RAPD扩增条带多态率最高（93.4％），AFLP单组引物扩增多态性条带最多，平均为23.25条，但多态率较低。RAPD与SRAP分析聚类结果与基于形态学分类基本一致，但AFLP分析聚类结果与基于形态学分类有差异。研究结果表明萝卜各种质之间的遗传多样性与肉质根皮色及地理来源有关，采用多种分子标记综合分析萝卜品种之间的遗传关系更加准确、可靠。萝卜花药与花粉培养研究中采用了KT、NAA及2.4-D三种激素，5个萝卜品种探讨了不同激素组合与基因型对萝卜花药及小孢子愈伤诱导与分化的影响，结果显示2.4-D为0.2mg·L^-1或KT4 mg·L^-1+NAA0.2 mg·L^-1时对萝卜愈伤诱导效果较好，并且KT4 mg·L^-1+NAA0.2 mg·L^-1的激素组合愈伤生长较快，NAA增至2 mg·L^-1时根分化明显，但芽的分化较困难。5个品种中NAU-Yb-03与NAU-Dy-13-03对激素反应较为敏感。在萝卜多倍体诱导研究中采用0.3％秋水仙素与12℃条件下采用0.1％的秋水仙素对春化与未春化的NAU-Dy-13-03分别进行不同处理次数与时间的生长点涂抹与浸种处理。通过观察及倍性鉴定发现幼苗生长点涂抹处理在刚露真叶期没有效果，二叶一心期植株成活率为100％且处理四次效果较好，加倍率达50％，表明选择适当的萝卜苗龄与处理次数对生长点涂抹处理有重要影响。而浸种处理不同时间表现出梯度效应，处理3～6.5h下胚轴增粗，并能抽伸出真叶，效果较好。形态学观察表明叶片形态与加倍效应有很大关系，在萝卜倍性鉴定中有重要作用。根据本文的研究结果，认为利用分子标记技术进行萝卜遗传多样性研究，种质资源鉴定切实可行的，但是每一种标记由于在基因组中分布的不同，从而导致遗传进化存在差异，因此在分析结果上可能会存在差异，综合利用多种分子标记研究较为可靠。本研究中得出的最佳激素组合将为萝卜花药的进一步完善提供重要的基础。多倍体诱导体系的初步研究为萝卜多倍体在实践中的应用提供了可能。更多还原

【Abstract】 The application of the combined technology system consists of molecular markers,anther culture and polyploid inducing to breeding of radish will be great helpful fordevelopment of radish which depends on the collection, analysis and innovation of radishgermplasm.In this paper, the analysis of the genetic diversity and identification of 17 radishes wasconducted with 107 RAPD primers, 4 pair of AFLP primes and 13 pair of SRAP primersand the possibility of employing the molecular markers to the research of geneticrelationship of radishes was discussed. The result showed that RAPD marker was thehighest polymorphic （93.4％） in three kinds of markers. The average 23.25 fragmentsamplified with AFLP prime combinations were obtained but with relatively lowerpolymorphic ratio. The dendrograms based on RAPD and SRAP data showed highconsistency with the classification according to the morphological traits, while the result ofAFLP markers analysis showed some difference. It could be concluded that the geneticdiversity of radish was associated with the color of fresh root skin and origin. Moreover, asfor revealing genetic relationships of different accessions in radish ,the data from severalkinds of markers analysis were more reliable than that from only one type of markeranalysis.In the part of anther and isolated microspores culture, three kinds of hormoneincluding KT, NAA and 2.4-D were employed to analyze the affecting of differentcombinations of hormone and genetypes on the initiation and differentiation of callus. Theresult showed that 0.2mg·L^-12.4-D and 4mg·L^-1KT in combination with 0.2 mg·L^-1NAAwere effective for callus initiation. The combination of 4mg·L^-1KT with 0.2 mg·L^-1NAAresulted in fast growth of cellus and the foot differentiation of cellus was easy when theconcentration of NAA was 2 mg·L^-1, but the bud differentiation was very difficult on anycondition. NAU-Yb-03 and NAU-Dy-13-03 were more sensitive to hormone than theothers in five varieties.In the research of polyploid induction of radish, two methods were applied to induce the polyploid of radish. First, the buds of vernalized seedlings of NAU-Dy-13-03 were dippedat first and third-leaves period in 0.3％colchicine solution with different times. Second, theunvernalized seeds of its were immerged in 0.1％colchicine solution at 12℃with differentlong time. The result showed that 100 percent of the treated materials survived and 50percent of polyploid-induced ratio was obtained when the materials were treated four timesin third-leaf period which was better than in first-leaf period. So it could be concluded thatboth the age of seedlings and times of treating were important for the effect of ployploidinduction of radish by buds-dipped method. The inducing degree with different long timeshowed gradual effect in seeds-immerged method, and hypocotyls became wider andmorphological character of leaves became more like that of polyploid obtained in the firstmethod when the treating time was from 3h to 6.5h. As for polyploid identification,morphologic character of leaves may play an important role.We can concluded that it is available to identify the germplasm of radish withmolecular markers, but it is more reliable with several markers than only one, since thedifferent distribution of various markers in the genome results in their genetic difference ofevolution. At the same time, the combination of hormone obtained in the research makes agood base of anther culture of radish, and the research of polyploid inducing makes itpossible to apply the polyploidy varieties of radish in the practice.更多还原