Studies On Numerical Taxonomy And SCoT Marker Of Rose Natural Hybrids

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.