【作者】 王玉；

【导师】 赵兰勇；

【作者基本信息】 山东农业大学 ， 风景园林学， 2015， 硕士

【摘要】 玫瑰(Rosa rugosa)以其重要的园林造景价值和经济价值,成为蔷薇属重要的落叶观赏灌木和必不可少的种质资源。本研究以‘紫枝’玫瑰(Rosa rugosa’Zizhi’)‘北京单白’(Rosa rugosa’Beijingdanbai’)、‘唐红’(Rosa rugosa’Tanghong’)3个玫瑰品种,探究玫瑰叶片基因组DNA的最佳提取方法。以数量分类和SCoT分子标记两种方法,在形态和分子两个层面上探讨玫瑰40个自然杂交后代的分类鉴定、遗传多样性及亲缘关系等,为玫瑰种质资源的保护和利用及新品种选育提供科学依据。主要研究结论如下:1.玫瑰基因组DNA的适宜提取方法改良CTAB法和试剂盒法。改良CTAB法适用于样品量少,试验时间充足,经费条件不高的条件下;试剂盒法适用于经费宽裕,样品材料珍稀的条件下。各实验室应根据自身具体情况选择合适的提取方法。2.玫瑰自然杂交后代的数量分类2.1 Q型聚类将40个玫瑰自然杂交后代分为5类(Z为杂交后代简称):第一类:Z1,Z15,Z36,Z39,Z2,Z5,Z7,Z8,Z19,Z20,Z4,Z3,Z9,Z6,Z32,Z29,Z30,Z33,Z18,Z24;第二类:Z10,Z17,Z14,Z21,Z13,Z16;第三类:Z12,Z38,Z40,Z35,Z37,Z22,Z28,Z23,Z31,Z25,Z26,Z27;Z11和Z34分别为第四类和第五类。分类依据依次为花色、花瓣数量、花梗刺的着生位置等。2.2 R型聚类将37个玫瑰性状指标分为5类,分别集中体现玫瑰花器官、小叶、托叶、枝刺等特征信息。性状选择基本正确,适于玫瑰自然杂交后代的数量分类与鉴别。2.3主成分分析通过利用SPSS17.0软件中的主成分分析,分析37个性状,得到9个主成分,累计贡献率达81.324%,可代表原始性状的大部分信息。第1主成分贡献率最大,特征向量绝对值较大的性状有花直径、花丝长、花药长、雄蕊长、雄蕊瓣化与否、雄蕊群宽、花蕾长/宽、花梗刺着生部位、花瓣白斑有无、花瓣数及小枝刺等性状,说明第1主成分是由若干实际测量性状组成的一个综合指标,包含区分40个玫瑰自然杂交后代的主要形态特征。以第1主成分为主,适当选择其它8个主成分,即可实现对玫瑰自然杂交后代的准确分类。3.玫瑰自然杂交后代的scot分子标记研究3.1最适反应体系在正交试验和单因素试验的基础上得到玫瑰最适的scot-pcr反应体系,即20μl的反应体系中,含有2.00μl10×pcrbuffer,1.50ng/μl模板dna,2.00mmol/lmg2+,0.35mmol/ldntps、0.70μmol/l引物,0.50utaq酶。各因素对该反应体系的影响大小依次为:mg2+>taq酶>模板dna>引物>dntps。扩增程序为94℃预变性5min;94℃变性1min,50℃退火1min,72℃复性2min,35次循环;72℃延伸10min,4℃保存。该体系也适用于玫瑰自然杂交后代的scot-pcr反应。3.2引物多态性从36条引物中筛选出能稳定扩增的20条引物,利用这20条引物对40个玫瑰自然杂交后代进行扩增反应,共得到426条带,多态性条带为379,每个引物平均产生的多态性条带数为18.96,多态性比率88.97%。有效等位位点数在1.3730~1.7574之间,均值为1.5963;nei’s遗传多样性指数(h)在0.2346~0.4103之间,均值为0.3406;shannon信息指数(i)在0.3590~0.5879之间,均值为0.5011。3.3遗传距离玫瑰40个自然杂交后代间的遗传相似系数介于0.5141~0.9859之间,平均值为0.6659。其中z4和z5之间的遗传相似系数最大为0.9859,遗传距离最小为0.0142,两者亲缘关系最近;z9和z34之间的遗传相似系数最小为0.5141,遗传距离最大为0.6653,两者亲缘关系最远。3.4分类体系根据scot分析结果,采用upgma法构建聚类树状图,聚类分析结果将40个玫瑰自然杂交后代分为5类,第一类:z1,z2,z4,z5,z6,z7,z8,z15,z18,z19,z20,z29,z33,z24,z30,z32,z39,z36,z3,z9;第二类:z21,z22,z28,z31,z23,z25,z26,z27,z35,z12,z37,z38,z40;第三类:z11;第四类:z10,z17,z13,z16,z14;第五类:z34。分类依据基本以花色、花瓣数等花器官性状为主。4.不同分类方法得到的分类体系一致玫瑰40个自然杂交后代经数量分类和scot分子标记两种分类方法均得到5个分类体系,其中前3类与以往研究结果所得蔷薇系、月季系和玫瑰系相一致,另有其他2个类型。两种方法的聚类结果分析认为:数量分类和scot分子标记对玫瑰40个自然杂交后代的分类结果基本一致,都将其分为5个类群,且各类群中包含的玫瑰自然杂交后代大致相同。经互相验证,方法选择可靠,结论基本准确。更多还原

【Abstract】 Rosa rugosa is an improtant kind of deciduous ornamental shrub of genus Rosa which has great landscape and ecnomic value,it is also the gerplasm resource in breeding.3 rosa cultivars,Rosa rugosa‘Zizhimeigui’,‘Beijingdanbai’,‘Tanghong’were analysised to explore the best genomic DNA extraction method from the leaves.40 rose natural hybirds were studied on calssification and identification,genetic diversity,phylogenetic relationships and other aspects with numerical taxonomy and SCo T marker.The analysis above provided a scientific basis for the further study on molecular biology level of rose later,the proteciton and utilizaton of rose germplasm resource and new rose varieties’ breeding. The main results were as follows: 1 Suitable extraction method for genomic DNA of Rosa rugosa.Modified CTAB method and DNA minprep kit method were the most suitable genomic DNA extraction methods of rose leaves.The former could be applied with few samples,adequate time or insufficient funds,the later could be applied with enough funds and rare samples. A appropriate method could be chosen according to the actual conditions of the labs. 2. Analysis on numerical taxonomy of rose natural hybirds 2.1 Q type cluster40 rose natural hybirds were divided into 5 groups by Q cluster analysis.The first group : Z1,Z15,Z36,Z39,Z2,Z5,Z7,Z8,Z19,Z20,Z4,Z3,Z9,Z6,Z32,Z29,Z30,Z33,Z18,Z24;the second group: Z10,Z17,Z14,Z21,Z13,Z16;the third group: Z12,Z38,Z40,Z35,Z37,Z22,Z28,Z23,Z31,Z25,Z26,Z27;Z11 and Z34 was the fourth and fifth group.The basis of classification above was petal color,number of petal and the growth position of stalk thom of rose natural hybirds in turn. 2.2 R type cluster37 rose characters were divided into 5 groups by R cluster analysis.Each group reflected the flower,leaves,stipile and thorn characters.The characters selected were right basiclly,and sutiable for the numerical taxonomy analysis and identification of rose natural hybirds. 2.3 Principal component analysis37 rose characters were analysised by principla component analysis method with SPSS17.0, a total of 9 principal components were obtained and the cumulative contribution rate was 81.324%.The first principal component had the maximum contribution rate,the characteristics with larger characteristic values were the diameter of flower,the length of filament,the length of anther,the length of stamen,petaliform stamen(yes/no),the width of stamens,the length/width of flower bud,stalk thom,spot on petal(yes/no),the number of petal and twig thorn,the result indicating that the first principla component was a comprehensive index composed of several acrual observed value traits,it contained the main morphological characteristics of 40 rose natural hybirds.Selected other 8 principla components appropriately based on the selection of the principal component 1,the accurate classification of rose natural hybirds could be achieved. 3. Analysis on SCo T marker of rose natural hybirds 3.1 The optimal reaction systemOrthogonal design experiment and single factor experiment were conducted to establish the reaction system for SCo T-PCR of Rosa rugosa.The total 20μL reaction volume included 2.00μL of 10×PCR buffer,1.50ng/μL of DNA template,2.00mmol/L of Mg2+,0.35 mmol/L of d NTPs、0.70μmol/L of primer,0.50 U of Taq enzyme.The influences of each factor on the PCR-reaction system was Mg2+>Taq enzyme>DNA template>primer>d NTPs.Amplification program was: an initial denaturation step at 94℃ for 5 min, followed by 35 cycles of 94℃ for 1 min, 50℃ for 1 min, and 72℃ for 2 min;the final extension at 72℃ was held for 10 min and stored at 4℃. 3.2 Primer polymorphism20 primers were selected from 36 primers,which were applied to study the genetic diversity and relationships of the materials.20 primers produced 426 bands of which 379 were polymorphic,the percentage of polymorphic bands was 88.97%,each primer produced 18.96 polymorphic bands;the Effective number of allele was 1.3730~1.7574 with an average of 1.5963;the Nei’s genetic diversity(H) was 0.2346~0.4103 with an average of 0.3406;and the Shannon’s information index(I) was 0.3590~0.5879 with an average of 0.5011. 3.3 Genetic distanceThe Nei’s genetic similarity coefficient(GS) ranged from 0.5141~0.9859 with an average of 0.6659.The genetic similarity coefficient between Z4 and Z5 was the maximum of 0.9859 and the genetic distance was the smallest 0.0142,which were the closest relationship; the genetic similarity coefficient between Z9 and Z34 was the minimum of 0.5141 and the genetic distance was the largest 0.6653,which were the farthest genetic relationship. 3.4 Classification systemA dendrogram was constructed based on SCo T data using UPGMA cluster method.40 rose natural hybirds were classified into 5 groups,which was mainly based on petal color,petal number and other floral organ characters. The first group : Z1,Z2,Z4,Z5,Z6,Z7,Z8,Z15,Z18,Z19,Z20,Z29,Z33,Z24,Z30,Z32,Z39,Z36,Z3,Z9;the second group: Z21,Z22,Z28,Z31,Z23,Z25,Z26,Z27,Z35,Z12,Z37,Z38,Z40;the third group: Z11; the fourth group : Z10,Z17,Z13,Z16,Z14;the fifth group:Z34. 4. Classification system were the same with different classification methods40 rose natural hybrids were both dived into 5 groups with numerical taxonomy and SCo T marker.The first three groups were consistent with the results of previous studies :’qiangwei’ type,’yueji’ type and ‘meigui’ type,while with two other types.From the clustering results with the two methods we could preliminarily think that:the classification result of numerical taxonomy and SCo T marker were basically the same,which both divided the 40 rose natural hybirds into 5 groups,and each group included the same strains. It proved that the two methods selected were reliable and the conclusion was baisclly accurate by verifing the results obtained mutually.更多还原