【作者】 姬文秀；

【导师】 李虎林；

【作者基本信息】 延边大学 ， 作物遗传育种， 2009， 硕士

【摘要】 大字杜鹃（Rhododendron schlippenbachin Maxim.）杜鹃亚属,野生种。其植株展枝优美,花大色艳,为较好的观姿、观花植物,大字杜鹃在吉林省仅分布在珲春市境内,数量十分稀少。本研究对大字杜鹃进行了细胞学研究,为揭示其种间关系、起源和演化提供必要的资料;同时利用RAPD技术对大字杜鹃群体间和群体内进行遗传多样性的分析及亲缘关系的鉴定,旨在进一步建立大字杜鹃核心种质以及更好地利用其资源奠定基础,为我国大字杜鹃育种工作提供参考。研究结果如下:1、通过对大字杜鹃进行核型分析得到大字杜鹃体细胞染色体数2n=28,染色体基数n=14,染色体核型公式为2n=2x=28=12m+16sm,核型为2B,臂比变幅为1.08～2.59,相对长度变幅在10.44%～4.40%,着丝点指数变幅为27.84%～48.51%,平均值为38.13%,核型不对称系数为61.17%。2、以改进的CTAB法提取大字杜鹃叶片基因组DNA,分别研究了模板DNA浓度,引物浓度,dNTP浓度,Taq DNA聚合酶量及镁离子浓度等因素对大字杜鹃RAPD反应结果的影响。通过对各因子的组合比较,建立了大字杜鹃RAPD反应的优化体系,即总反应体积为25μL,其中包括10×Taq酶配套缓冲液2.5μL,模板DNA浓度为50ng/μL,引物浓度为0.5μmol/L,dNTP浓度为0.15mmol/L,Taq DNA聚合酶为1U,Mg²⁺浓度为2.0mmol/L,其余用重蒸馏水补足。扩增程序为94℃预变性5min,94℃变性1min,38℃退火40s,72℃延伸2min,共41个循环,72℃延伸7min。3、采用RAPD技术对大字杜鹃6个天然群体进行了遗传多样性分析,从160个随机引物中筛选出了重复性好、有丰富多态性的8个引物,共产生176条带,其中多态性带153条,占86.93%。根据RAPD二元数据结果,计算出了6个群体间的遗传距离和相似系数,并根据UPGMA法进行了聚类分析,建立了6个大字杜鹃群体间的聚类分析树状图。结果表明:6个大字杜鹃群体可以分成3类,第1类为中国延边珲春敬信北坡和敬信南坡,第2类为朝鲜琵琶岛北山、牡蛎泡和泽思岭,第3类为朝鲜琵琶岛海边,初步鉴定了大字杜鹃不同种群之间的亲缘关系。4、采用RAPD技术对延边州珲春敬信南坡大字杜鹃群体内23个个体进行了遗传多样性分析,筛选出了5个引物,共产生73条带,其中多态性带68条,占93.15%。根据RAPD二元数据结果,计算出了23个个体间的遗传距离和相似系数,根据UPGMA方法进行聚类分析,建立了同一群体内23个大字杜鹃个体间的聚类分析树状图。结果表明:23个大字杜鹃个体可以分为3类,分别为:第1类包括1、2、11、3、8、9、12、19、18、21、23、4,第2类包括10、20、13、14、15、17,第3类为5、6、7、16、22。更多还原

【Abstract】 Rhododendron schlippenbachin Maxim.is wild species and belongs to Rhododendron subgenus.It is a fine scenic plant because of the nice posture and the beautiful flower,but the amount of Rh.schlippenbachin Maxim.is too little to find nowhere except in Hunchun city in Jilin province.The research took the karyotype analysis for the Rh.schlippenbachin Maxim.,which is necessary to reveal the relation of interspecies,the origin and the evolution,and the genetic diversity was researched among not only 6 population of Rhododendron schlippenbachin Maxim.but also individuals in a population on RAPD.The results are following:1.The chromosome number of somatic cell 2n=28,the cardinal number n=14,the karyotype formula is 2n=2x=28=12m+16sm,the karyotype is 2B,the range of arms ratio is 1.08～2.59,the range of relative length is 10.44%～4.40%,the range of kinetochore exponent is 27.84%～48.51%,its average is 38.13%,and the karyotype dissymmetry coefficient is 61.17%.2.The genomic DNA was extracted from Rhododendron schlippenbachin Maxim.by means of advanced CTAB method,we studied that the influences of the concentration of template DNA,primer,dNTP,Taq DNA polymerase and Mg²⁺ on RAPD analysis procedure.We also established the optimization system of RAPD on the base of the comparation among various influence factor groups:The total volume 25.L,comprising of 10×Taq buffer（2.5.L）,template DNA concentration 50ng/.L,the primer concentration 0.5.mol/L,dNTP 0.15mmol/L,Taq DNA polymerase 1U,Mg²⁺ concentration 2.0 mmol/L and the supplementary ddH₂O.The extended program was: 94℃predenaturation 5min,94℃denaturation 1 min,38℃annealing 40 s,72℃extension 2 min,41 cycles in all,72℃extension 7 min finally.3.The experiment researched the genetic diversity for 6 population of Rhododendron schlippenbachin Maxim.with RAPD,we filtered 8 primers which worked well on repeatability and polymorphism,the total number of band is 176,among which the number of polyme- rphisic band is 153,occupied with 86.93%.Cluster anaysis were taken with the method of UPMGA in software named NTSYS,and finally the dendrogram has been established.As a result,when the genetic distance is 0.45,the 6 population of Rh.schleppenbachin Maxim.were classified into three parts:the first are from the southern and northernslope Jingxin Hunchun Yanbian city in China;the second is from northern mountain of pipa island,ZSL and QP in DPRK;the third is from the slope on the seaside of pipa island in DPRK.4.The experiment also researched the genetic diversity for 23 of Rh.schlippenbachin Maxim.in one population with RAPD,we filtered 5 primers,the total number of band is 73,among which the number of polymorphisic band is 68,occupied with 93.15%. Cluster anaysis were taken with the method of UPMGA in software named NTSYS,and finally the dendrogram has been established.As a result,when the genetic distance is 0.37,individuls of the same Rh.schleppenbachin Maxim.population were classified into three parts:the first are NO.1,2,11,3,8,9,12,19,18,21,23,4,the second are NO. 10,20,13,14,15,17,and the third is NO.5,6,7,16,22.更多还原