| Dendrobium thyrsiflorum Rchb.f is a traditional Chinese herb of Dendrobium, Orchidaceae. The stems of D. thyrsiflorum can be processed into "Huangcao Shihu,' an important class of Traditional Chinese Medicine with good curative effect. It has been used as a tonic medicine to enhance the immune function, nourish Yin and clear away heat-evil, benefit the stomach and promote the production of body fluid, moisten the lung and relieve a cough. D. thyrsiflorum is also used as an ornamental plant because of its graceful flowers. However, the wide usage of D. thyrsiflorum in medicine and horticulture has caused the over-exploitation of its natural resources over the past few decades, leading to a substantial decrease in its stock.Analysis of the genetic diversity and structure of an endangered species is a necessary prerequisite for conservation as it reflects the status and survival potential of populations. At present, the population genetics of this species remains unknown, which makes it difficult to implement effective conservation strategies. In this paper, simple sequence repeat (SSR) marker was employed to investigate genetic diversity and population genetic structure of this species for clarifying the causes of its endangerment and defining potential conservation strategies. Accordingly, elucidating the origin of D. thyrsiflorum is critical for the quality control of commercially available "Huangcao Shihu." In addition, once the geographic origin of collected D. thyrsiflorum individuals is validated, effective measures can be taken to prohibit its over-exploitation and protect its wild resources.The identification of the geographical origin of D. thyrsiflorum is vital for preserving its natural resource and ensuring the quality of "Huangcao Shihu." In order to identify the origin of D. thyrsiflorum on Chinese herbal medicine market,14 D. thyrsiflorum-specific microsatellite markers were developed in this study and were used to investigate the genetic diversity and differentiation of 5 wild D. thyrsiflorum populations consisting of 136 individuals collected from China, Thailand, India, Myanmar, and Laos, respectively. Microsatellite marker analysis coupled with the new statistical approach (Bayesian method) was used to identify the geographic origin of 12 commercial samples of "Huangcao Shihu.'In this research, microsatellite DNA was isolated from D. thyrsiflorum genome through the method of enrichment by magnetic beads.24 primer pairs were designed from micro satellites. With the genomic DNA of 5 fresh D. thyrsiflorum samples collected from China, PCR was performed to test the effectiveness of these primers in consistent amplification reflected in the size of products. Of the 24 primer pairs,14 pairs were finally chosen for analysis of the genetic characteristics of the 14 SSR loci of D. thyrsiflorum.14 microsatellite loci developed in the present study were used to explore genetic diversity and population structure using genotypic data from 136 individuals collected at five different localities (China, Myanmar, Laos, Thailand and India). On the species level, the detected number of alleles was 136 in 14 loci. The average number of alleles (Na) and effective number of alleles (Ne) were 9.71 and 6.83, respectively. The detected number of alleles per locus ranged from 5.8 to 12 with an average of 8.0. The averages of the shannon diversity index and expected heterozygosities across loci were 2.02 and 0.85, respectively. By the population level, the effective number of alleles of 5 populations ranged from 4.45 to 6.83. The range of expected heterozygosity (He) among populations was from 0.76 to 0.85, suggesting a high level of genetic diversity in D. thyrsiflorum natural populatons. The coefficient of genetic differentiation (Fst) was ranging from 0.06 to 0.22, and the mean value of Fst was 0.12, which indicated that 12% of the genetic variation occurred among populations. That means a relatively high level of genetic differentiation among populations despite the existence of most genetic variation within populations.Based on the microsatellite data of 5 D. thyrsiflorum populations collected from China, Thailand, India, Myanmar, and Laos, respectively, the software (GENECLASS 2.0 and STRUCTURE 2.2) based on the Bayesian Theory were applied to identify the origin of 12 commercial samples of "Huangcao Shihu." The results showed that four samples were found to originate from Myanmar, another four from Laos, two from China, one from Thailand, and one from India. The results suggest that Myanmar and Laos are the main geographical origins of D. thyrsiflorum on Chinese herbal market. This information implicates that the natural resources of D. thyrsiflorum in Myanmar and Laos may have been overly exploited, so it is urgent for these two countries to take feasible measures to protect it. |
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