Study On Systematic Of Taxonomy Of Some Composite Plants In ChangBai Mountain Area By RAPD And SRAP Markers

Compositae is a family that is the china seed plants that it is the most popular, genetic relationship and evolution of complex system in the world. It has the high theoretical research value and economic value, and has the wide attention of the world scholars. There are more than 150 wild species at the ChangBai Mountain Area that is precious genetic resources, and these kinds wide distribution, habitat complex, good adaptability. This study were analyzed using RAPD and SRAP molecular markers by cluster analysis and genetic diversity of 16 wild compositae materials at the ChangBai Mountain Area, Which provide theoretical basis to reveal the relationship between the origin, evolution; Using this technique for part of wild Compositae were analyzed. RAPD and SRAP technology used in the relationship between species, feasibility study for further research on the relationship between the various plant species provide more accurate the molecules evidence. The main results are following:1. The genomic DNA was extracted from Compositae plants by means of advanced CTAB methed, and purification processing. According to the experiment of references this study choose the influences of the concentration of Mg2+, dNTP, template DNA, primer, and Taq DNA polymerases factors for optimizing the factor, and using orthogonal design to optimize the RAPD reaction system. The final RAPD reaction conditions was:2.5μL 10×PCR buffer,40 ng template DNA, Mg2+ 2.0 mmol/L, dNTP 2.0 mmol/L,primer 0.4μmol/L,Taq DNA polymerase 1U, in a total of 25μL reaction solution. The extended program was:94℃pre degeneration 5 min,94℃degeneration 1 min,40℃annealing 1 min, 72℃extension 2 min,40 cycles in all,72℃extension 10 min.2. The study researched the genetic diversity for 16 copies of wild Compositae with RAPD, we screened 16 primers from 100 UBC random primer which worked well on repeatability and polymorphism, the total number of band is 239, among which the number of all polymorphism band, various primers polymorphism of several different bands, polymorphism bands in 9-20, polymorphism is 100%.Cluster analysis were taken with the method of UPMGA in software named NTSYS, and finally the dendrogram has been established. According to this map, the GS of 16 samples of Compositae Plants is 0.5348-0.7720. Among them, when GS is 0.66 the 16 samples of Compositae Plants were clustered into 4 classes. The first one is Ixeris denticutata (Houtt.)Stebb,Aretium lappa L.,Cirsium pendulum Fisch.ex DC,Cisium setosum (wiid.)Bieb,Bidens parviflora Willd,Helianthus tuberosus L.,Siegesbeckia pubescens Makino,Erigeron Canadensis L.,Xanthium strumarium L.,Scorzonera albicauis Bunge,Leibnizia anandria (L.)Turcz,Bidens tripatita L.; The second one is Lactuca indica L.; The third one is Sonchus oleraceus L.,Kalimeis indica; The forth one is Artemisia argyi Lev.et Vant. When the GS is 0.70, the first one was clustered into 8 small classes, that are①Ixeris denticutata (Houtt.)Stebb②Aretium lappa L.,Cirsium pendulum Fisch.ex DC③Cisium setosum (wiid.)Bieb,Bidens parviflora Willd,Helianthus tuberosus L.④Siegesbeckia pubescens Makino,Erigeron Canadensis L.⑤Xanthium strumarium L.⑥Scorzonera albicauis Bunge⑦Sonchus oleraceus L.⑧Cisium setosum (wiid.)Bieb.3. Orthogonal design and uniform design were used to obtain optimum scheme on reaction system for sequence-related amplified polymorphism (SRAP). And the optimum reaction systems of the two designs were compared. Results showed that both orthogonal design and uniform design could be used for SRAP. But compared with the orthogonal design, uniform design could avoid blindness and quickly obtained the optimum reaction system for SRAP with clear bands and well stability. The optimized SRAP-PCR system was:2.5μL 10×PCR buffer,30 ng template DNA, Mg2+ 2.5 mmol/L,dNTP1.5 mmol/L,primer 0.3μmol/L,Taq DNA polymerase 1U, in a total of 25μL reaction solution. The results showed that Orthogonal design were feasible to apply SRAP optimizing the reaction system. The extended program was:94℃pre degeneration 4 min,94℃degeneration 45 s, 35℃annealing 1 min,72℃extension 90 s,5 cycles; 94℃degeneration 45s,50℃annealing 1 min,72℃extension 90 s,35 cycles;72℃extension 10 min.4. The experiment researched the genetic diversity for 16 copies of wild Compositae with SRAP, we screened 10 primers from 60 SRAP primers which worked well on repeatability and polymorphism, each SRAP primer combinations of clear band is 10-16.10 pair SRAP primers produced all band is 117, the average of producing bands 11.7 per primer, among which the number of polymorphism band is 116, the average of producing polymorphism bands 11.6 per primer, polymorphism is 99.14%.Cluster analysis were taken with the method of UPMGA in software named NTSYS. According to this map, the GS of 16 samples of Compositae Plants is 0.5128-0.7436. Among them, when GS is 0.635 the 16 samples of Compositae Plants were clustered into 3 classes. The first one is Ixeris denticutata (Houtt.)Stebb,Bidens tripatita L.,Bidens parviflora Willd;The second one is Aretium lappa L.,Xanthium strumarium L.,Siegesbeckia pubescens Makino,Cisium setosum (wiid.)Bieb,Helianthus tuberosus L.,Cirsium pendulum Fisch.ex DC,Artemisia argyi Lev.et Vant,Leibnizia anandria (L.)Turcz,Kalimeis indica,Erigeron Canadensis L.; The third one is Scorzonera albicauis Bunge,Sonchus oleraceus L.,Lactuca indica L.. When the GS is 0.68, the second one was clustered into 4 small classes, that are (1) Aretium lappa L.; (2) Xanthium strumarium L.,Siegesbeckia pubescens Makino,Cisium setosum (wiid.)Bieb,Helianthus tuberosus L.,Cirsium pendulum Fisch.ex DC,Artemisia argyi Lev.et Vant; (3) Leibnizia anandria (L.)Turcz; (4) Kalimeis indica,Erigeron Canadensis L.; When the GS is 0.73, the small second one was clustered into 3 small classes, that are①Xanthium strumarium L.,Siegesbeckia pubescens Makino②Cisium setosum (wiid.)Bie,Helianthus tuberosus L.③Cirsium pendulum Fisch.ex DC,Artemisia argyi Lev. et Vant.5. According to the results of calculation RAPD and SRAP data of genetic similarity coefficient method, by UPGMA cluster analysis, The dendrogram will be clustered RAPD and SRAP cluster results and the morphology of the traditional classification comparison and analysis, the results is that RAPD marker technology in the classification of within the division between the traditional and the morphology of classification, can only be applied in individual species of classification; The SRAP cluster analysis compared with the traditional classification, the basic is consistent, comprehensive clustering figure, the clustering results obtained with RAPD and SRAP cluster results, the results compared with traditional classification, closer to the morphological indirect evidence can also more than RAPD analysis SRAP analysis is stable and accurate.