【作者】 张旭；

【导师】 周兰英；

【作者基本信息】 四川农业大学 ， 园林植物与观赏园艺， 2010， 硕士

【摘要】 牡丹在长期的进化和栽培历史中,由于本身的变异、自然杂交、栽培环境不同等形成了不同的品种类型,使得牡丹的分类鉴定难度加大。本试验以彭州地区种植的部分栽培品种为材料,应用RAPD分子标记技术,对39个牡丹品种的遗传多样性进行了随机扩增多态性DNA(RAPD)研究,通过NTSYSpc(2.10版)软件对RAPD的扩增谱带进行分析,进行牡丹品种的分子鉴定,探讨牡丹品种之间的亲缘关系。其主要的试验结果如下：(1)采用改进的CATB法提取牡丹幼嫩叶片的DNA,分别对其进行紫外吸收检测、电泳检测和RAPD检测,结果表明：DNA完整性好,RAPD扩增带清晰,A260/A280的值在1.8-2.0之间,满足RAPD反应的要求。(2)通过对模板DNA、引物、TaqDNA聚合酶、dNTP和Mg2+浓度的优化试验及对PCR反应程序的筛选,确定了本研究的适宜反应体系和反应程序。反应体系：模板DNA 1μL(20ng/μL),10×PCR缓冲液2μL,随机引物0.8μL(2.5μmol/L)、dNTPs0.8μL(2.5mmol/μL)、MgCl21.6μL(25mmol/L), Taq-DNA聚合酶0.25μL(5U/μL),双蒸水13.55μL,总反应体系20μL。反应程序为：94℃预变性5min,94℃变性1min,36℃退火1min,72℃延伸1.5min,共40个循环,最后72℃延伸10 min,4℃保存。(3)利用对100个引物进行筛选所得到的8个引物对39个牡丹样品进行RAPD分析,共检测到139个位点,其中118个多态位点,多态位点百分率达到84.89%。平均每个引物获得17.38个扩增位点,其中具有多态性的位点为14.75个。各品种间多态位点百分率变化范围为79.36-88.19%,其中多态位点百分比最高的是引物S30,最低的是引物S85。(4)利用RAPD技术对39份牡丹材料进行分类研究,通过聚类分析,估算遗传距离,建立树状图,当相异系数为0.46时,39个牡丹品种明显聚为两大类,本土牡丹品种为第1大类,日本牡丹品种为第2大类。第1类包括35个品种,又可以分为2个亚类：第1亚类包括31个品种均为中原牡丹品种；第2亚类包括4个品种,均为彭州当地牡丹品种。由此结果可以看出,多数来源地相同的品种表现出较为密切的亲缘关系。更多还原

【Abstract】 Owing of the variation with in the speeies, easily natuer eross between speeies and wide generation, dieffrent planting enviromnent in the world, many speeies populations and Variety groups of penoy have been forming in the long history of penoy evolution and culturing, which make taxonomy and identification of penoy difficult and naming confusing. The study was undertaken to identify penoy material of several grapes of pengzhou area in sichuan provice. Though molecular markers technology of RAPD to studies the genetic diversity of 39 series penoys. The aim of the studies on the amplification patterns of RAPD got by NTSYS pc2.10e is to identificate and taxonomy penoy,study linkage distance.The main results as follows:(1)The methods-CTAB method were used to extract genomic DNA from leaves of grape. The DNA samples of the three methods were tested by spectrophotometer,agarose gelelectrophoresis and RAPD. The result was that the DNA integrality extract with those methods was good,the DNA trap was distinct, the amplified band of the RAPD is clear, the number of A260/A280 lied between 1.8and 2.0,which is in accordance with reaction requirements of RAPD.(2)The different PCR program and the ingredients in RAPD reation system such as DNA and primer,Taq DNA polymerase, dNTP,Mg2+, were sceened by different concentrations. Suitable reaction system and program of the RAPD for penoy genomic DNA was settled. The volumn of amplified reation was DNA 1μL (20ng/μL), 10×PCR buffer 2μL, Random primer 0.8μL(2.5μmol/L)、dNTPs0.8μL(2.5mmol/μL)、MgCl21.6μL(25mmol/L), Taq-DNA Polymerase 0.25μL(5U/μL), Double distilled water 13.55μL,total reaction 20μL. The reaction program was devised for 45 cycles,each with 94℃Pre-denaturation for 5 min,94℃denaturation for 1 min,36℃annealing for 1 min, 72℃extension for 2 min, at last 72℃elongation for 10 min,4℃for preservation.(3) 20 primers which are the better of 20 primers in the experiment are used to analyze the genetic diversity of 39 series penoy using RAPD markers.139 sites were found. Among which there are 118 polymorphic loci. The percentage of polymorphic loci is 84.89%. The average number DNA bands produced by each primer was 17.38, and theat of polymorphic loci was 14.75. The percentage of polymorphic loci among different penoy varies from 79.36% to 88.19%. The percentage of polymorphic loci of primers including S30 is the highest as the same time the lowest one is S85.Those above results showed that the genetic diversity of 39 accessions was high.(4) 39 kinds of Peony are researched in classification by RAPD technology, according to the result of cluster analysis, estimate the genetic distance, set up tree chart, when the dissimilarity coefficient is 0.46,39 kinds of Peony are obviously gathered into 2 kinds. Local Peony belongs to the first kind, while Japanese Peony belongs to the second kind. The first kind includs 35 varieties, which are divided into 2 subgroups, the first subgroup includs 31 varieties that are zhongyuan Peony. The second subgroup includs 4 varieties that are local Peony of Pengzhou. The conclusion based on the result bofore are that the variety of Peony coming from same place can show a close relatives relationship.更多还原