Study Of ISSR Markers And ITS Sequences Analysis Of Dendrobium

This paper describes the optimized method for the total DNA isolation of Dendrobium species. A DNA extraction method for the PCR of Dendrobium species was found and ISSR markers were used for the first time in the identification of Dendrobium species. We established and optimized the ISSR-PCR procedure to fit the Dendrobium species. It was used to identify and distinguish 12 species of Dendrobium and 8 populations of D. qfficinale. On the other hand, the result of ITS region sequencing proved that the wild D. qfficinale found in Fuyang, Zhejiang Province was veracious. Result are follows:1. The optimized CTAB method was seccessfully used to extract the polysaccharide-rich genomic DNA of Dendrobium. This method is widely used at present in our lab with satisfactory purity and quantity of the extract. The extracted DNA is suitable for ISSR-PCR.2. The optimized ISSR-PCR system of Dendrobium was: 25 uL reaction system contained 50 ng template, 1 uM primer, 2.0 mM Mg2+, 200 uM dNTP, 2% formamide and 1U Taq polymerase. The PCR parameter was: 94# pre-denature for 7 min; 94#denature for 30 sec, 52'C annealing for 45 sec, 72'C extended for 2 min, 45cycles; 72# extended for 7 min, the product of PCR was kept at 4#. By using the optimized conditions, we could obtain repeatable results and legible electrophoretic patterns.3. The 21 primers, except PI, could produce amplification bands at different degrees. 13 of them had excellent amplification patterns in 12 species and they formed 144 amplification bands in total. 8 of the 13 primers formed 109 bands, and each of them was polymorphic. Consequently, polymorphism detected by the ISSR-PCR method among Dendrobium species was very high. Each of the primers, P9, S7, S8, S9 and S10 each could identify the above mentioned 12 species alone.4. 5 primers were used to distinguish the 8 populations of D. officinale. 4 of the primers formed 47 amplification bands and 42 of them were polymophic, so the polymophic /total band ratio was 89.4%. Another primer, P7, didn't have polymophic band, but all the populations of D. officinale had the same clear band in the 700 bp region. For other species of Dendrobiwn, the primer P7 had slightly different bands. Primer S9 and S10 could distinguish all the 8 populations of D. officinale respectively.5. Results of the ISSR-PCR showed that samples of D. officinale from Fujian and Hunan which flowers in light purple had the electrophoretic pattern consistent with other populations' of D. officinale. Consequently, according to the electrophoretic pattern, we could confirm that this sample was really D. officinale.6. According to the ITS sequence comparison with the GeneBank's, identities were 95% for the D. officinale from Yunnan and 99% for the one from Fuyang, Zhejiang respectively. Thus, the molecular data proved that the D. officinale from Fuyang, Zhejiang was veracious.