| Dendrobium nobile Lindl. (Orchidaceae) is an endangered and endemic herb mainly distributed in China.’Huangcao’, which was made of the stems of D. nobile was listed as one of the best restorative foods in Shen Long Ben Cao Jing. It has long been used as famous and expensive health food in many Asian countries. However, habitat destruction and over exploitation are threatening its survival in recent years, and it has become an endangered species. Consequently, there are other kinds of adulteration products often mixed with D. nobile in the health care product. There is a pressing need to strengthen the protection among wild population of D. nobile, and develop an accurate, sensitive and rapid method for authenticating ’Huangcao’ from other adulteration.Germplasm preservation of D. nobile was taken in this study,and we proposed a method based on a special treatment to improve the seed setting rate of D. nobile and applied for a patent. The special treatment is containing Ca(NO3)2, H3BO3, sucrose, urea, gibberellin and aqueous solution of 2,4-D. The step of method mainly involved: the selection of parents, the pollination time, artificial pollination, bagging treatment, to obtain the seed of D. nobile and the seeds were hormone free tissue culture. Such a kind of special treatment was good at improving the seed setting rate of D. nobile. This method improved the plant production obviously and was easy to operate and master, which could be applied in large-scale artificial cultivation.Fifty-three novel microsatellite markers were isolated by a modified biotin-streptavidin capture method and tested the genetic diversity of 4 populations of D. nobile in Yunnan, Guizhou and Guangxi Province. Ten pairs of microsatellite markers for D. nobile showed a great polymorphism, the PIC ranged from 0.55 to 0.85 (PIC> 0.50). The 10 microsatellite markers revealed considerable genetic diversity of D. nobile, which can be used to evaluate the conservation efficiently, and will be helpful in management decision-making of D. nobile conservation.The conservation genetics research towards D. nobile was carry out in this study. We picked 9 polymorphic microsatellite loci to reveal the genetic diversity and population differentiation of 7 wild D. nobile populations in the southwest China (Hainan Island, the border between Yunnan and Burma, Yunnan Province, Guizhou Province, Guangxi Province, Sichuan Province). Several genetic statistics analysis used in this study to discuss the effect of different evolutionary processes in shaping the genetic composition of D. nobile populations, and to suggest a strategy for the conservation of D. nobile in southwest China. After scoring the alleles for the 9 loci, a total of 148 alleles was detected in the 102 D. nobile samples. The number of alleles per locus ranged from 12 to 32, with an average of 16.4 alleles per locus. The observed heterozygosity (Ho) varied from 0.503 to 0.794 (mean 0.695), and expected heterozygosities (HE) ranged from 0.623 to 0.909 (mean 0.74). The 9 SSR loci exhibited relatively high PIC values ranging from 0.546 to 0.86 (mean 0.675), and all markers were highly informative (PIC> 0.50) for further genetic diversity studies in D. nobile. The level of genetic diversity and genetic variation tended to be lower in the Hainan Island population. Accordingly, the D. nobile population distributed on Hainan Island needs particular protection, like in situ conservation and restoration of habitat; and individuals in Hainan Island with rare alleles need to be conserved with top priority, those individuals with rare alleles and the most common alleles also should be concerned.In order to provide essential data for plant identification and comparative studies in Dendrobium and Orchidaceae.The complete chloroplast genome of D. nobile was sequenced, and compared the difference of complete cp genome among the Dendrobium whose chloroplast genomes were already known. Furthermore, we develop identification markers and microsatellite marker in the cp genome (cpSSR) of D. nobile. Accordingly, the size of Dendrobiums’cp genome and IR region are very conservative. The chloroplast genome of D. nobile has a total length of 150,793 bp and comprised a large single copy region (LSC,84,939 bp), a small single copy region (SSC,13,310 bp), and two inverted repeat regions (IR,26,272 bp). By performing the diagnostic real-time PCR assay based on SYBR Green I, a 109 bp and a 127 bp fragment was specifically amplified from the chloroplast gene ycfl and trnL-trnF intergenic spacer of D. nobile, respectively. The results showed distinct melting peaks which indicated that primer pair SDyf/SDyr and SDtf/SDtr is specific in identifying D. nobile from other adulterants. The established system can be used in discrimination of’Huangcao’from other adulterants rapidly, unequivocally and widely. |
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