【作者】 邱长玉；

【导师】 周瑞阳； 高国庆；

【作者基本信息】 广西大学 ， 作物遗传育种， 2008， 硕士

【摘要】 茉莉花（Jasminum sambac（L.）Atiton）木樨科素馨属多年生灌木,起源于波斯湾一带,种质资源十分丰富。但长期以来,对茉莉花资源的分类和利用,都是采用林奈和恩格勒的形态特征分类法,往往有些种、品种和品系在外貌和形态特征上十分相似,但亲缘关系和遗传特性却相差巨大,使得茉莉花的分类地位及亲缘关系一直存在着较多的争议,并给茉莉花的新品种培育和资源利用带来困难。本试验以广西区域内广泛收集的茉莉花品种为试材,应用ISSR分子标记技术,探讨茉莉花的分类和品种之间亲缘关系的研究。试验结果表明:1、通过对SDS、CTAB和改良CTAB法三种基因组DNA提取方法的比较,从纯度、产率等方面考虑,认为改良CTAB法较为适合茉莉花基因组DNA的提取。采用该方法提取的茉莉花基因组DNA,经电泳检测,主带清晰,无降解,分光光度计检测,A₂₆₀/A₂₈₀的值在1.81—1.84之间,符合PCR反应的要求。2、通过对Taq DNA聚合酶、dNTP、模板DNA、Primer不同浓度的优化试验及对PCR反应程序的筛选,确定了合适的反应体系和反应程序,从而建立了茉莉花的ISSR分析技术体系。在20μl反应体系中,模板DNA 75ng,Primer 5μmol,rTaq polymerase 1 U,dNTP 4 mmol,buffer(含Mg²⁺)2ul。反应程序为:预变性,94℃5min;变性,94℃1min;退火,X℃1min;延伸,72℃2min;40个循环,72℃延伸10min,4℃保存。3、利用筛选得到的10条ISSR引物对全部茉莉花材料进行PCR扩增,30份扦插苗得到70个位点,其中多态性位点数为34个,多态位点百分率为48.57%;扩增135份实生苗得到70个位点,其中多态性位点数为42个,多态位点百分率为60%;扩增24份实生苗得到66个位点,其中多态性位点数为44个,多态位点百分率为65.7%。扩增片段长度分布在100bp-2000bp之间,平均每个引物能扩出7条带。4、运用NTSYS软件,采用Dice相似系数分析ISSR反应结果,得到了24个品种间的亲缘关系树状图。以0.77作为相似系数的分界点,24个实生苗样品通过基因组ISSR分成2大类:花蕾较多的为一类,另一类在相似系数0.84的水平将其余的23个茉莉花材料分为4个品种群:群Ⅰ:叶型基本相同的;群Ⅱ:分枝少,花期晚;群Ⅲ:无分枝,花期晚;群Ⅳ:无分枝,花蕾少。该结果与传统的分类结论基本一致。5、利用ISSR技术可以对基因组的所有序列直接分析,不受环境的影响,在分子水平上利用ISSR技术对供试茉莉花品种（系）进行遗传多样性和亲缘关系鉴定,结果非常可靠,是种质资源研究中快速、可靠、有效的新技术,能为杂交育种亲本组配提供有价值的分子水平上的信息。更多还原

【Abstract】 Jasminum sambac（L.）Atiton is sweetclover Branch Jasminum,which originated in Persian Gulf.and its variety resource is very abundant.The utilization and classification of Jasmine germplasm resources were adopted by the botany classification system of linnaean or Engler for a long time. There are arguements on the genetic relationship and classification among the native varieties of Jasmine for many years,which had made grate trouble in breeding new variety and utilizating resource.As some species,cultivars or strains are much similar in the appearance and morphological characters, while their relationships of heredity and relative were very different. Therefore,in this study we analyzed the classification and genetic relationship among 189 cultivars of Jasmine by ISSR-PCR.The main result of this research is as following:1.Three DNA extraction methods of CTAB,SDS and modified CTAB were studied.The results showed that the modified CTAB method was the best to extract Jasmine genomic DNA based on the purity and yield of DNA. The products（bands）were clear and undegradation by agarose gel electrophoresis.The values of A₂₆₀/A₂₈₀were between 1.81 and 1.84 detected by U/V spectrophotometer.This method could get good purity DNA and was qualified to apply to ISSR-PCR analysis.2.The different PCR programs and the ingredients of ISSR reaction system such as Taq DNA Polymerase,dNTP,DNA and primers were screened by different concentrations.Suitable reaction systems and programs of the ISSR analysis for Jasmine genomic DNA were designed.The following was the 20μL reaction system with DNA template 75ng,Primer 5μmol,rTaq polymerase 1 U,dNTP 4 mmol,buffer(including Mg²⁺)2ul.The procedure was that predenature at 94℃for 5min,denature at 94℃for 1min,anneal at X℃for 1min,elongate at 72℃for 2min,cycling number at 40,holding at 72℃for 10min,and keeping final products at 4℃.3.10 ISSR primers screened from 100 ISSR primers were utilized for the amplification reaction of Jasmine template DNA by PCR reaction.A total of 70 bands were obtained by amplification of the polymorphic primers of 30 cuttings,among which 34 bands were found to be polymorphic.The percentage of polymorphic bands was 48.57%.A total of 70 bands were obtained by amplification of the polymorphic primers of 135 seedings,among which 42 bands were found to be polymorphic.The percentage of polymorphic bands was 60%.A total of 66 bands were obtained by amplification of the polymorphic primers of 24 seedings,among which 44 polymorphic bands were found to be polymorphic.The percentage of polymorphic bands was 65.7%.Every primer could amplify 7 bands averagely, and the size of band amplified was between 100bp and 2000bp.4.The dendrogram was obtained by the UPGMA clustering method.24 Jasmine cultivars could be clustered into two groups according to the dendrogram.Of there,one group has only a variety,that is with more buds. The other was a big group,which could be divided into four sub-groups,the first subgroup has the same shape of leaf,the second subgroup owns the character of a few branches and buding late,the third subgroup with few branch and buding late,the fourthly subgroup with few branch and buds.5.All DNA template can be effectively analyzed by ISSR method,which is not influenced by environmental conditions.It was quite reliable that the genetic diversity and relationship of 189 Jasmine cultivars were analyzed by ISSR method at the molecular level.ISSR-PCR analysis is a relatively new technology,which is very simple,reliable and highly effective in studying the Jasmine germplasm resources.And it could provide some valuable information for parent combinations on the hybridization breeding at the molecular level.更多还原