Phylogenetic Relationship Of Eriocaulon Six Sections By RAPD And ITS Sequences Analysis

The high intraspecific variability combined within the limited interspecific differences makes the taxonomy of Eriocaulon Linn, very difficult. Up to now, the studies on Eriocaulaceae through molecular technique mainly have focused on the phylogenetic relationship between Eriocaulaceae and its relative families, but little is known about the intrageneric variability of Eriocaulon. Based on the RAPD system construction of Eriocaulon plants, molecular technique (RAPD and ITS sequences) combining with morphology was used to analysis the phylogenetic realationship of 6 sections and genetic diversity of 9 species of Eriocaulon in order to validate the classification existed, which would provide basis for the further study on the confirmation of the phylogenetic position of Eriocaulaceae and Eriocaulon.Comparative analyses have been made in order to select the suitable method of DNA extraction and the best RAPD system of Eriocaulon plants in this study. The resultsshowed that the best optimum concentration range of template DNA, Mg2+, dNTP andprimer were 20-40 ng, 1.5-2 mmol L-1 , 80-200 umol L-1 and 30-40 ng, respectively, ina 25 uL reaction volume. And the best conditon of PCR reaction is 94 C for 4 min, forty-five cycles of 94 C for 15 s, 35 C for 45 s and 72 C for 90 s, and then 72 C for 4 min.The results of RAPD showed that 107 bands (92.24 %) behave polymorphic in 116 RAPD bands amplified by 12 random primers out of 70. Using UPGMA in NTSYS computer programes, the data of 116 RAPD bands were used to generate Nei's similarity coefficients and genetic distances and to construct a dendrogram.ITS sequences of 5 species in Eriocaulon were analysised in this study, indicating that the ITS sequences of Eriocaulon plants were not completely identical. The size of ITS 1 and ITS2 of these 5 species was 171-306 bp and 184-288bp, respectively. G+C content ofITS1 and ITS2 was about 38.24-55.04 % and 36.46-57.02 %, respectively. Using PAUP 4.0 10b software, Juncus dregeanus (Juncaceae) as outgroup, the aligned sequences were analysized to construct the striction consensus tree, in which tree length, CI and RI were1180, 0.7983 and 0.3479, respectively.The analysis of RAPD molecular marker and ITS Sequences indicated that there was extensive genetic diversity in the species of E. cinereum R. Brown, and among the species of Eriocaulon. It was suggested that E. kunmingense Z.X.Zhang and E. cinereum R.Brown should be put into one section. E. buergerianum Koernicke and E. alpestre Koernicke were relatives, which was confirmed in this study, as the case was for E. nepalense Proscott ex Bongard and E. oryzetorum Martius. Sect. Simplices Fyson and Sect. Apoda Z.X.Zhang was sister group, another was for Sect. Anisopetalae Fyson and Sect. Disepala Z.X.Zhang. Additionally, Sect. Heterochiton and Sect. Leucantherae Fyson were relatively far from the other sections on phylogenetic relationship, and these two sections were far from each other.