Analysis Of The Phylogenetic Placement Of Pohlia And Rhodobryum By RAPD In Bryaceae(MUSCI)

The molecular systematics of Bryaceae is new focus in the study on moss. RAPD has been widely used for molecular systematics, and it's an important technique in phylogeny analysis. The placement of Pohlia, the status of Rhodobryum genera and the phylogenetic relationship of several generas within Bryaceae are conflictive. In this study, the optimum random amplified polymorphic DNA (RAPD) reaction system for Bryaceae was built, then the RAPD profile of Bryaceae was construct. The phylogenetic relationships of the mosses in virtue of the RAPD profile in this study were explored.After the comparative method was performed, the genomic DNA extracted by rapid kit with good integrality and purity is enough to perform RAPD. The single factor level experiment and orthogonal test were used to analyze the factors influencing RAPD and the optimum reaction was established. The results showed that the suitable program was one cycle at 94℃for 7 min, 40 cycles at 94℃for 1min, 36℃for 1min, 72℃for 2 min and one cycle at 72℃for 7min. In the RAPD system of Bryaceae the optimal concentration of template DNA was 2ng/μl, primer 0.4μmol/L, dNTP 0.2mmol/L, Mg2+ 2mmol/L, Taq DNA polymerase 1.25U, total volum 25μl. Several DNA samples were selected to choose the compatible primers. The primers were selected by which stabilized products with great polymorphism were acquired. Thirty-five primers were selected from 250 primers by amplifing template DNA from sample Y1, Y2 and Y3 under the optimum reaction system.The selected primers were applied to amplify all DNA samples, and twenty-six primers were used to create the RAPD profile finally. Differential bands of RAPD appeared clearly among samples. 198 bands were amplified with these primers, in which 181 bands were polymorphic. The percentage of polymorphic bands was 91.41% and the mass weight is between 200bp~5000bp.The polymorphism information was acquired from gel imaging system. Phylogenetic analysis was carried out by using Paup4.0b8. Maximum parsimony analyses (MP), neighbour-joining analyses (NJ) and unweighted pair group method using arithmetic average analyses (UPGMA) from collected information. Phylogenetic trees were created and comparative analysis was performed. The phylogenetic relationships is different from traditional classification, supporting the results from molecular analyses.The result from phylogenetic trees created respectively by MP, NJ and UPGMA indicates that Bryaceae and Bryum are not monophyletic. The definition of some groups of Bryaceae and Bryum is doubtful in traditional classification based on morphological data. The analyses approves that Pohlia is more closely related to Mniaceae than Bryaceae, which supports placement of Pohlia in Mniaceae from Bryaceae based on DNA sequence data. Rhodobryum appears as sister of Bryum, which suggests that Rhodobryum ought to rank in Bryum genera. It may not be suitable to recognize Rhodobryum as subgeneras in Bryum.The polymorphic profile could reflect the polymorphism of the taxa and related taxa. RAPD can provide some molecular proof for Bryaceae's phylogeny. There is no need to study deeply on Phylogenetic relationships of Bryaceae.