The Study On Relationship And Genetic Diversity Of Rhododendron

In this study,the relationship and genetic diversity were investigated using Rhododendron materials from the southwest mountainous region of Sichuan where the altitude is 2020-3435m and the major methods and results are as follows.The mathematic classification of morphologic character was studied.44 morphologic characters including 18 dualistic characters,13 multi-characters and 13 quantitative characters,were selected and analyzed using SPSS10.0.Squared Euclidean distance coefficient was used in case clustering and Pearson correlation was used in variable clustering by within-groups linkage.The 46 species of Rhododendron were divided into two caboodles:one was lepidote and the other was evergreen by case clustering.The different groups were formed under two caboodles and the groups were similar to the traditional classification except R.calophytum and R.asterochnoum.The results of variable clustering showed that the characters were decided in different groups.In principal component analyses(PCA),the accumulative contribution of the fifth principal component just up to 70%while the first three principal components was up to 60.9%, which showed that some important characters can be used in classification of Rhododendron,such as squama,gland,hair and the size of leaves,flowers and fruits. However,the number of stamen,hair on sepal,size of sepal,color of crown,hair on lower silk and hair on crown etc have a small contribution on classification.The results of PCA was in according with that in case clustering,which reflected that we should pay more attention to choose character in classifying.Pollen grains of 26 wild Rhododendron in southwest mountain region of Sichuan were observed by scanning electron microscope(SEM),in which 21 species were reported for the first time.They belonged to 3 subgenera(Subgonus Rhododendron,Subgenus Hymenanthes,Subgenus Pseudorhodorastrum),4 sections and 12 subsections.All of them are tetrahedron with tetrad construction.Pollen grains of Rhododendron were spherical or subspherical with tricolporate and few of them with four colporates.There was unequal visein thread on the surface.The size of tetrad and the pollen exine sculpture of them were different in SEM,but no conspicuous difference by the colpus and proximal exine.The diference of pollen exine sculptur and length and width of germinal apertures did not fit for sorting the subgenera,while the diameter of tetrahedron could provide proof for further taxonomy.In the same subgenera,the morphological data of pollen could assist to identify the section and subsect.It was not suitable to identify some certain species in Rhododendron only by morphological data of pollen,however,it might be used for identification of some species in certain section and subsection.Essential factors affecting the result of Rhododendron RAPD-PCR were studied with orthogonal design.And the optimal RAPD-PCR were built as follow:DNA4μL (20ng/μL);10×PCR buffer 2μL;Taq E 1.2μL(2.5U/μL);primer 0.3μL(0.5OD),dNTPs1.1μL(2.5mmol/L),MGCl₂2.5μL(25mmol/L),in 20μL reaction solution. Amplification reactions was performed in a 96-well thermocycler(Eppendorf Authorized Thermal Cycler PCR) programmed as follows:an initial denaturizing at 94℃for 3min followed by 45 cycles of 1min at 94℃,39s at 34℃,1min at 72℃,and finally extended at 72℃for 10 min.49 samples of 43 wild Rhododendron species from the southwest mountainous region in Sichuan were studied by using RAPD,including 25 samples of 23 species in Subgenus Hymenanthes,18 samples of 18 species in Subgenus Rhododendron and 6 samples of 2 species in Subgenus Pseudorhodorastru,according to Sleumer's classification system. Their genetic diversity,relationship and phylogeny were analyzed.They were clustered by UPGMA.The results showed that:1) 407 DNA fragments were amplified by 24 primers, among which 399 DNA fragments were polymorphic,up to 98.03%.Dendrogram obtained by UPGMA implied the genetic diversity of Rhododendron;2) Their coefficient of genetic similarity(GS) was ranging 0.25～0.82,the average GS was 0.4631;3) The division by RAPD was consistent with the 3 subgenus classification system by morphological characters.49 samples were divided into 3 groups by cluster analysis based on the GS:the first group contained 25 samples belonging to Subgenus Hymenanthes;the second group contained 18 samples belonging to Subgenus Rhododendron,and the third group consisted of 6 samples of Subgenus Pseudorhodorastrum.The first two groups were clustered at GS 0.40 then gathered with the third group at GS 0.37.Subgenus Hymenanthes had a closer relationship with Subgenus Rhododendron than with Subgenus Pseudorhodorastru,so Subgenus Pseudorhodorastru should not be mergered into Subgenus Rhododendron.