【作者】 朱红霞；

【导师】 袁涛；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2004， 硕士

【摘要】 牡丹、芍药为中国的传统名花，在中国园林和花文化中有着重要的地位和影响。为了有效的鉴别品种、研究品种亲缘关系、有效地保护利用这一优良的种质资源，并为牡丹、芍药品种国际登录和新品种保护提供分子水平的品种鉴定技术，本研究在摸索适用于牡丹、芍药的荧光法AFLP分子标记技术体系的基础上，初步建立了牡丹、芍药部分品种DNA指纹图谱。得到以下主要结论： (1) 建立并优化了适宜于牡丹、芍药的AFLP反应体系，并在样品采集、DNA提取、酶切连接、两次扩增以及显带等实验过程优化的基础上，得到了稳定、清晰、分辨率较高的指纹谱带。 (2) 利用内切酶Mse Ⅰ-EcoR Ⅰ的引物组合，初步建立了5个牡丹品种、6个芍药品种的DNA指纹图谱。牡丹的5对引物组合得到214条多态性条带，多态百分率为80.4％，芍药的4对引物组合得到179条多态性条带，多态百分率为81.5％。表明AFLP确实是揭示多态性的强有力的工具。所有供试引物的鉴别效率均为100％，供试的11个品种均有其特异(具有或缺失)的谱带，证明AFLP分析是目前鉴定品种较有效的方法之一。 (3) 运用AFLP标记进行牡丹、芍药指纹图谱绘制时，选择性碱基引物组合E-AGG／M-CAG、E-ACT／M-CAT分别为牡丹、芍药较理想的引物组合。 (4) 鉴别亲缘关系相近的品种时，有时一对引物组合不足以检测出差异，应选用两对或更多对引物，并要注意参考和结合传统分类方法。 (5) 牡丹品种‘冠世墨玉’的形成可能是‘黑花魁’、‘烟笼紫珠盘’经天然杂交后，基因发生突变的结果；也可能在其形成过程中有其它品种参与。有待于扩大样品量后进一步研究。由遗传距离看，芍药品种‘Primevere’与‘种生粉’的遗传距离最近，为0.6667，与它的相似性最大。由于‘Primevere’的亲缘关系不清，供试材料的有限，本文只是根据所的结果从分子水平上进行分类。 (6) AFLP在植物鉴别和系统分类方面，虽然有强大的说服力，但它并不排斥其它的方法。应综合形态学、孢粉学、细胞学、同工酶分析等多方面的证据，才能得到较完美的解释。摘要 以上研究为牡丹、芍药品种鉴定、优良品种选育、新品种保护初步奠定了分子标记研究的理论基础，同时，为进一步构建牡丹、芍药遗传连锁图谱、分子标记辅助育种、重要基因的图位克隆等提供了可供参考的AFLP反应体系。更多还原

【Abstract】 Peaonia suffruticosa and P. lactiflora are Chinese famous traditional plant, playing an important role in Chinese gardening and people’s cultural life. The objectives of this study are to develop superior cultivars, and to effectively conserve and utilize the superior genetic resources, to identify new cultivars for registration. DNA fingerprinting were established for typical varieties, based on the AFLP techniques and the DNA fingerprinting, the identification of tree peaonies and herbaceous peonies cultivars were studied with use of SSR software. The main results were as follows:(1) An AFLP system suitable for P. suffruticosa and P. lactiflora was developed, clear and high resolution bands were obtained with appropriate handlings in sample collection, DNA extraction, restriction-ligase reaction, and double amplification as well as staining techniques throughout the experiment process.(2) With use of Mse I - EcoR I digest and primers, fingerprinting of the 11 samples were obtained. P. suffruticosa ,With 5 combinations of primers, we obtained 257 bands among which 214 were polymorphic ones with 80.4% percentage of polymorphism being; Paeonia lactiflora , With 4 combinations of primers, we obtained 215 bands among which 179 were polymorphic ones with an average identifying percentage of 95.87%. The result showed that AFLP was truly a useful tool to test polymorphism among closely-related cultivars. 9 combinations of primers distinguished all tested cultivars completely, 11 cultivars were found to have cultivar-specific(having or missing)AFLP fingerprinting. The result showed that AFLP was one of the most effective method in identifying cultivars at present.(3)The primer E-AGG/ M-CAG was proved to be ideal combinations of primers for AFLP analysis of P. suffruticosa . The primer E-ACT/ M-CAT are better ones for P. lactiflora.(4)In the analysis among similar cultivars, singer pair of primer was not enough to distinguish their difference sometimes. We should select two or more pairs of primers to analysis and combine some other traditional methods.(5)’Guan Shi Mo Yu’ is possible a natural hybrid of ’Yan Long Zi Zhu Pan’ and ’Hei Hua kui’ and is also likely be affected by other cultivars. ’Primevere’and ’Zhong Sheng Fen ’have higher genetic similarity,genetic distance is 0.6667.(6)AFLP is an effective method for plant identification and classification. Combination of AFLP techniques with morphological, palynologicah catonological and enzyme techniques is essential for variety identification.The preliminarily constructed fingerprinting provided useful information not only for developing superior cultivars and new variety right protection but also for conservationn and utilization the genetic resources. The improved systems could be used to construct standardized fingerprinting archive of genetic map, marker-assisted selection and map based cloning in peony . The results, therefore, will produce important effects on genetic improvement of peony at the molecular levels.更多还原