【作者】 唐晓敏；

【导师】 周立人； 李爱青； 曹德菊；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2003， 硕士

【摘要】 本试验进行了红麻11个常规栽培种和3个野生种的细胞学标记及RAPD分子标记的研究。通过建立14个品种的核型模式图在染色体水平上比较红麻不同品种间的遗传差异，并在探讨红麻种子基因组DNA提纯方法和优化PCR扩增反应体系的基础上，利用RAPD技术对红麻品种基因组DNA多态性进行研究，检测出各品种的特异性DNA谱带，并依据分子标记研究结果探索了栽培种与野生种的亲缘关系，为红麻种质资源的系统进化研究和开发利用提供了科学依据，同时也为红麻种质鉴定建立起一种快速、简便、实用的方法。通过以RAPD分子标记为主，细胞学标记为辅的红麻品种遗传差异和种质鉴定的研究分析，结果表明： 1．细胞学标记研究采用了稍加改进的F-BSG法获得了根尖细胞染色体分散、平展、分裂相多、随体清晰的染色体制片。核型分析结果表明14个品种的核型均为基本对称核型，但染色体的随体大小、数目等方面还存在一些差异。所以，红麻细胞学研究可以作为一种细胞学标记用于红麻不同基因型材料亲缘关系及种质鉴定的分析。 2．为了缩短时间，减少工作量，本试验探索出一种经济、快速、简便、能够用于RAPD分析的红麻种子基因组DNA提取新方法。针对红麻种子中富含蛋白质、脂肪和糖类等贮藏物质，可能还含有丰富的酚类物质及酚氧化酶等，在提取液中加入适量β-巯基乙醇，有效地防止氧化褐变，排除杂质的干扰。同时，采用氯仿-异戊醇代替酚抽提，避免了酚难去除带来的交叉污染等，获得了高浓度和高质量的基因组DNA，较好地用于PCR扩增，进行RAPD分析。 3．稳定的RAPD反应体系是进行RAPD分析的关键。通过实验优化建立了合适的RAPD反应体系。在25μl反应体系中含有：1×buffer，2.0mmol／LMgCl₂，200μmol／L dNTPs，0.6μmol／L随机引物，30ng基因组DNA，0.75U Taq酶。反应扩增程序为：94℃预变性5min，使模板DNA充分变性，然后进入下列温度循环：94℃变性30sec，38℃退火45sec，72℃延伸1min30sec，重复40个热循环，结束循环后72℃终延伸7min。按照优化的RAPD条件进行的重复实验，重现性较好，为红麻分子生物学研究提供参考依据。 4．采用RAPD技术对红麻14个品种进行基因组DNA多态性分析，从150个随机引物中筛选出了重复性好、有丰富多态性的99个引物，共产生766条带，其中多态性带347条，占45．3％，反映不同红麻品种间存在遗传差异。扩增片段长度大多数集中在0．2～1．skb之间。应用SAS软件，采用系统聚类法中的AverageLinkage Cluster Analysis对 GD进行遗传分类，构建树系图，将 14个红麻品种聚成七类，这同一般形态分类和己知的亲缘关系较吻合。从聚类结果中还可以看出，励海紫茎和元江红麻虽然原产地都是在云南，但两者的遗传差异较大，元江红麻是早熟特性，而励海紫茎是迟熟特性；NA 414尽管是野生种，但它同红引13 5的遗传差异为0刀7，亲缘关系比较近，它们在表型上也表现出一定的一致性。说明用RAPD方法对红麻进行分类和亲缘关系研究是有效的。 5．RAPD分析能检测出大量变异，通过比较各品种间的特异带或特征带的差异，可进行品种鉴别和纯度鉴定。利用14个引物扩增获得的14条RAPD特征带，作为一组 DNA指纹图谱可以区分所有供试的 14个红麻品种。其中，MX 247、UG 93、NA 414、塔什干、古巴8号上个品种分别在弓I物S153、S18。、S184、SZ、S186扩增时产生 S153－320hp、S18。－1543hp、S184－1100hp、SZ－1100hp、S186－980hp特征性条带，利用这些特征带可进行红麻品种的早期鉴定。 6．本研究的创新之处就在于首次将RAPD分子标记技术同细胞学标记技术相结合应用于红麻 11个常规栽培种和 3个野生种的遗传差异研究，探索品种间亲缘关系，并根据扩增出的特异性DNA谱带进行种质鉴定。研究表明不同品种的核型、随体大小及数目的差异与RAPD分析结果基本一致，RAPD分子标记和细胞学标记研究结合更能在一定程度上反映出品种的内在遗传差异。这些都表明RAPD技术辅之以细胞学标记技术检测出的DNA水平上的遗传差异，不仅有可能解决传统的红麻形态学分类方法所不能解诀的疑难问题，而且还可为红麻品种的早期鉴定提供分子指标。更多还原

【Abstract】 In this experimentation, random amplified polymorphic DNA (RAPD) technique and chromosome karyotype analysis were used to analyze the genetic relationship of eleven cultivars and three wild stirps of Hibiscus cannabinus L.. As to cytological markers, comparing karyotypes and finding heredity differences of them by establishing fourteen idiograms. The extraction methodology of genomic DNA of Hibiscus cannabinus L. was explored and the influential parameters were optimized. On the basis of them, RAPD was used to investigate the genetic polymorphisms among fourteen breeds. And the relations of them was to be researched, RAPD characteristic markers were to be found. RAPD molecular markers being main part assisting cytological markers, the analysis of heredity differences and breeds distinction were carried out. Results were as follows:1 .In studies of cytological markers, cells of chromosomes scattered, flatted, more split phases were obtained using modified F-BSG method. The results of study for karyotype showed that fourteen breeds had similar karyotypes, but they also had some differences among them. According to those we could identify them. Therefore this cytological research can be cytological markers used to analyze the relations and the breeds distinction of various genotypes of Hibiscus cannabinus L.2.To save time and reduce workload, a economical, rapid, simple, and applicable for RAPD analysis extraction methodology of seeds genomic DNA of Hibiscus cannabinus L. was explored. Aimed at much protein, grease, sugar and oxidized polyphenolic components in seeds of Hibiscus cannabinus L, it is a best way of adding suitable 3 -EM to lysis. Furthermore, high concentration and good quality seeds genomic DNA of Hibiscus cannabinus L. were obtainde using CIAA instead of propanone. The genomic DNA samples, prepared from seeds of Hibiscus cannabinus L. with aforementioned modified method were to be used as templates for polymerase chain reaction (PCR) and very suitable for RAPD analysis.3.Steady reaction system is the key point to RAPD analysis. The influentialfactors such as templates DNA, dNTPs, MgCb, primers, Taq polymerase and so on were studied and experimental parameters were optimized. They were as follows: 25 H 1 reaction system containing 1 X buffer, 2.0mmol/LMgCl2, 200 V- mol/L dNTPs, 0.6 V mol/L primers, 30ng template, 0.75U Taq polymerase. The optimal amplification program was as follows: 5min at 94 , followed by 94 for 30sec, 38 for 45sec, 72 for IminSOsec, 40 cycles and final extension time of 72 for 7min.This optimized experiment conditions could obtain reproducible results and provide good references to molecular biology.4.The genetic polymorphism of Hibiscus cannabinus Z.was investigated using random amplified polymorphism DNA marker, the detected materials included eleven cultivars and three wild stirps. Ninety-nine primers selected from one hundred and fifty primers amplified 766 RAPD fragments and 347 bands of them showed polymorphism, which occupied 45.3%. The length of most amplified fragments ranged from 0.2 to 1.5kb. The dendrogram was constructed by using SAS software and Average Linkage Cluster Analysis method based on their genetic differences calculated from RAPD data. It was concluded that fourteen breeds which have been tested in the research could be classified into seven groups. The map relatively accorded with morphological analysis and known relationships. It also showed that the genetic difference between Menghaizijing and Yuanjianghongma were comparatively farther, although they all originated from Yunnai. The genetic difference between NA 414 and Hongyinl35 were relatively closer, in spite of NA 414 is wild stirp. These fragments were efficiently used in the analysis of the relationship of the fourteen breeds.5.RAPD analysis can examine lots of variations. There were fourteen RAPD characteristic markers, which were detected as a group of DNA fingerprints to identify all the fourteen cultivars. Specific markers were found in five cultivars, that is MX 247, UG更多还原