DNA Barcoding Of Hainan Island Orchid Species Based On MatK And ITS Sequences

Orchidaceae is a plant groups that hard for identifying, especially in the non-fertile period, many species just have imperceptible difference among plants'morphologic, so it is impossible to distinguish them by the tradition classification. Because some of the Orchidaceae occur nature hybridization easily, there are many midle types and variation, the class bourn isn't very definitude, all of that lead to a difficulty in the classify and coordinate in genus and species. DNA Barcoding is a new technique which could assisted identification in taxonomy, and is the newest direction in molecule biology and taxonomy at present.In this study,158 wild orchids samples of Hainan were used as the material, Chloroplast matK gene and Ribosome ITS sequences were regarded as the DNA Barcoding to distinguish and appraise the wild orchidacae of Hainan. The Main results were as follows:(1) matK and ITS achieved the ideal DNA Barcode demand of candidate for fragment, could be used for DNA Barcoding research of Hainan wild Orchidaceae. Research results showed that matK's effective length was about 800bp, ITS sequence was about 600bp, both of their lengths conformed to the DNA Barcod requirement. MatK and ITS existed many information points,320bp and 434bp respectively, and contained enough biological information. In the effective materials, separate identification effectiveness of matK and ITS reached 96.90%and 81.08%respectively.(2) The DNA Barcoding technology system of Hainan wild Orchidacae was found Based on the matK gene and ITS sequence. Choose, and the PCR system was ajusted by the primers of matK4(390f/1326r) and ITS2 (AB101f/AB102r), the results showed that the ITS's amplication difficulty could be solved by adding PCR enhancer to the reaction system. ITS and matK were 100%amplified in the 158 samples. Acoording K2P was choose for genetic distance analysis, Wilconxon Signed Rank Text and the NJ phylogenetic tree analysis, the DNA Barcoding analysis method was integrated.(3) The Orchidacae genomic DNA extraction method named improved CTAB method was established which was a broad spectrum, simple and trace method. The genomic DNA of 150 orchidacae samples (involving 4 subfamilies,70 genus)were extraced by this method, the result showed that the improved CTAB method could remove the phenols, polysaccharide, protein and other impurities, The length of DNA were about 23kb,theA26o/A280 were between 1.6 to 2.0, the DNA concentration were between 158 to 508 ng/μ1, could satisfy the requirement of follow-up study.(4) The NJ phylogenetic tree was constructed based on the matK and ITS sequence, could assistant identification the Hainan wild Orchidacae effectively, and had a higher identified rate. Combining NJ phylogenetic tree and the morphological data,7 of the 17 unidentification species were distinguished to species, and the other 10 to genus successfully.(5) 145 matK sequences and 151 ITS sequences were obtained from the Hainan wild Orchidacae, which can be used for constructing the DNA Barcoding data bank.102 matK gene sequences and 89 ITS sequences were not logined in the Genebank.