Huoshan Dendrobium Stem Molecular Identification And Genetic Diversity Evaluation

Dendrobium huoshanenese Tang et Cheng, commonly known as "Mihu", is a rare and endangered perennial herbs that can only be found in Dabieshan District in Anhui province. It is one of the most important medical plants of the Dendrobium family. Most of them are growing in the crack of cliffs or trees. Over-exploited by people who covet the high medicinal value of D. huoshanense, it is facing the situation of becoming extinct. The sterm of D. huoshanense can be made into a famous Traditional Chinese Medicine (TCM) called "Huodou" which is worth70-100thousand yuan per kilogram. In recent years, some conscienceless medical merchant usually adulterate D. huoshanense with other less valued similar species to make huge profits. Therefore, it is deadly in need of analyzing genetic diversity as well as some efficient solutions to identify D.huoshanense from other similar species.This research focuses on three aspects as follows:(1) Microsatellite markers of Dendrobium thyrsiflorum are used to analyze it’s generality in D.huoshanense. The genetic diversity within D.huoshanense turn out to be0.661.(2) An identification test of D. huoshanense and9other species of Dendrobium was performed with specific primer which was designed according to the specific loci of nrDNA ITS sequence. The forward primer HS-JB01S based on96th loci of ITS1sequence, meanwhile, the reverse primer HS-JB01X based on474th loci of ITS2sequence. Only the DNA template of D. huoshanense can be revealed when the annealing temperature moves to66℃.(3) Two pairs of specific primers (HS_RT2and HS_RT6) which had the best amplification efficiency in D.huoshanense, were designed based on ITS sequences of60Dendrobium species. Therefore, a rapid and precise method, combining SYBR Green Ⅱ—based on real-time PCR, was established. By performing the real-time PCR with HS_RT2, results showed that the average cycle threshold (Ct) value of D. huoshanense was14.3, while the value of other species were33.57±0.74. Similarly, with HS_RT6, results showed that the Ct value of D. huoshanense was14.72, while the value of others were33.31±0.69. By means of ststistics analysis, there was statistical difference between D. huoshanense and other species (P<0.001).In this study, a DNA extraction method was established to reduce the high content of polysaccharide, which would have bad influnce in DNA amplification of Dendrobium species as weel as established an efficient way to identify "Huodou" made from D. huoshanense from other "Fengdou" products.