【作者】 王宏；

【导师】 李周岐；

【作者基本信息】 西北农林科技大学 ， 林木遗传育种， 2007， 硕士

【摘要】 杜仲(Eucommia ulmoides Oliv.)属于杜仲科杜仲属,是一种以药用为主并具有多种用途植物,作为杜仲资源开发中一项十分重要的基础性工作,杜仲良种选育广受重视。近年来,杜仲许多优良品种(无性系)被陆续选育出来并用于生产栽培,但由于许多品种(无性系)间形态差异不大,因而品种识别较为困难。为了提供有效的杜仲品种鉴别方法,并研究品种之间的遗传差异,本文用RAPD技术对14个杜仲优良品种(无性系)进行了DNA指纹的建立和分析,主要研究结果如下:1.改进了杜仲基因组DNA提取方法,并对杜仲RAPD扩增反应程序进行了优化。用经过改良的CTAB法提取的基因组DNA可以满足RAPD扩增的要求。本试验最终优化反应体系为: 25μL反应体积中, 10×buffer 2.5μL,TaqDNA聚合酶1U ,随机引物(5μmol/L)2.0μL , MgCl2 (25mmol/L)2.0μL , dNTP (2.5mmol/L)2.0μL,模板2mg/L,补ddH2O至25μL。扩增程序为:94℃预变性5min,94℃变性30s,37℃退火30s,72℃延伸90s,35个循环,72℃延伸10min,4℃保持产物。2.对500条随机引物进行筛选,选出32条扩增条带清晰,多态性好的引物。并用这32条引物对14个杜仲品种(无性系)进行了扩增,得到扩增条带191条,其中多态性条带117条,多态位点百分率为61.26%。不同引物扩增的条带数在4-9之间,平均每个引物扩增5.96条。3.建立了14个杜仲品种(无性系)在引物S465、S480、S486三条引物下的DNA指纹图谱,可作为品种(无性系)鉴定的分子依据。4.对14个杜仲品种(无性系)的RAPD数据进行了相似系数计算和聚类分析,并建立了14个杜仲品种(无性系)的聚类树状图,品种之间的遗传相似系数分布在0.56-0.93之间,遗传系数均值为0.82。在遗传相似系数为0.82时,可将14个杜仲品种(无性系)划分为5大类别。更多还原

【Abstract】 Eucommia is a multipurpose tree species belonging to the order of eucommia family, as an extremely important basic work in the exploitation of resource, the breeding of Eucommia gets more attention than before. Recently, more and more cultivars (clones) which have good character was bred, and cultivated. But there is a little difference in characteristic between cultivars (clones), and make identifying more difficultly when meet a requirement, In order to provide a dependable technology for the identifying species, and study the difference in heredity, we established the fingerprinting of 14 cultivars (clones) by the RAPD technology and analyzed it. The main results are as below:1.Establish a improved method used to extract total DNA from the leaves of eucommia,tested and compared the reaction system and the cycle program. The total DNA extracted by the improved CTAB method, can get a good RAPD fingerprints. The reaction system used in this study were as follows: 10×buffer 2.5μL, Taq DNA polymerase 1U, MgCl2(25mmol/L) 2.0μL, dNTP(2.5mmol/L) 2.0μL, prime (5μmol/L)2.0μL, template DNA 2mg/L in 25μL reaction system. The PCR reaction program was: predenature at 94℃for 5 min, then 94℃30s, 37℃30s, 72℃90s, 35 cycles, and finally extended at 72℃for 10min, keep the PCR products at 4℃; 2.32 primers which has amplified and polymorphic bands had been selected from 500 random primers, the 32 primers are amplified for 14 cultivars, there are 191 amplified primers and 117 primers showing abundant polymorphisms, accounting for 61.26% .DNA bands are between 4 and 9 according to amplifying of different primers, the average is 5.96;3.The RAPD fingerprinting is constructed by the primers S465, S480 and S486. which can provides a references for cultivars(clones) identifying;4.14 cultivars(clones) are analyzed, including coefficient analysis and cluster analysis, and established the genetic dendrogram.the range of coefficient is between 0.56 and 0.93, average is 0.82,when coefficient was 0.82,the cultivars(clones) could be classified into five categories;更多还原