Molecular Identification And Genetic Analysis Of Cold-Resistant Rhododendron Resources On North China Based On DNA Barcodes And Numerical Taxonomy

DNA barcoding is a method for identifying and characterizing species of organisms using a short DNA fragment.DN A barcoding can identify species rapidly and accurately based on that the inter-specific divergence is greater than infra-specific divergence.N umerical taxonomy is a discipline founded in the 1950 s.It is an objective,quantitative and intuitive way to explore the genetic relationships among plant taxa.In order to investigate the phylogenetic relationships between the cold-resistant Rhododendron resources in North China and clarify evolutionary taxonomy of subgenus in genus Rhododendron,we analyzed DNA barcode ITS2 sequences and 7 cpDNA fragments from 11 plants of 6 species in genus Rhododendron.At the same time,cluster analysis was conducted on 22 species of cold-resistant Rhododendron species and their relative species in North China based on morphological taxonomy.The main results are as follows:(1)By searching the published ITS2 sequences in NCBI database,there are 92 species representing 9 subgenuses of Rhododendron ITS2 sequences that were analyzed to build the phylogenetic tree of Rhododendron using maximum parsimony methods and Cassiope fastigiata,Enkianthus quinqueflorus as outgroup.The result inferred that: Rhododendron is a polyphyletic group,and each subgenus evolutionary degree is balanced;the 6 species of plants are located in themajor branches of their own subgenus;the relationship of subg.Pseudazalea,subg.Pseudorhodorastrum and subg.Rhodorastrum are complex and their phylogenetic position are difficult to distinguish;subg.Pentanthera and subg.Therorhodion are located in the base of phylogenetic tree that are consistent with the results of morphological classification;the branch of the Sect.Choniastrum,Sect.Azaleastrum of Subg,Azaleastrum and Sect.Ponticum of Subg.Hymenanthes were strongly supported,while Sect.Ponticum distinction was not obvious at Subsection.(2)7 sequences can be amplified through the PCR reaction from the 13 barcodes.The all 7 DNA fragments were sequenced successfully which are accD、mat K、ndhJ、rbcL、rpoC、ycf5、rpoC1.The rbcL、mat K、rpoC1 have a good distinction effect;the acc D and rpoC need to combine with other barcodes for a better application results;and ndhJ and ycf5 are of little value in the classification of cold-resistant Rhododendron resources in North China.(3)Using SPSS 18.0 software,the 22 species of Rhododendron were clustered into 3 large branches and 1 single branches by means of N umerical Taxonomy based on morphological characters.The level of the distance coefficient =17 can be used as the dividing line of the subgenus,and it has a good effect on the northern cold-resistant Rhododendron and its relative species.But,it is hard to find the appropriate line to divide the subgenus level.(4)Not only the molecular clustering results,but also the morphological clustering results showed that the phylogenetic position of R.schlippenbachii can not be together with subg.Tsutsusi,so it may have independent system position,which needs further research.