Differentiation of Dendrobium Species used as ”Huangcao Shihu” by rDNA ITS Sequence Analysis

Abstract

The genus Dendrobium Sw. is composed of 74 species and two varieties in China, and 32 species carry the name ”Huangcao Shihu” on the herbal medicine market, making the identification of the origin of ”Huangcao Shihu” difficult for consumers. Here, the ITS regions were sequenced and evaluated to differentiate the 18 Dendrobium species used as ”Huangcao Shihu”. Diversity in DNA sequences among various species was found with the inter-specific sequence divergence ranging from 3.2 % to 37.9 % in ITS1 and 5.0 % to 26.6 % in ITS2. Moreover, the variations within species were very low, ranging in sequence divergence from 0 to 3.0 % in ITS1 and 0 to 4.0 % in ITS2. Therefore, these species could be easily distinguished at the DNA level. Furthermore, based on the divergent ITS regions, five pairs of species-specific primers were designed and used for the rapid PCR identification of five Dendrobium species listed in the Chinese Pharmacopoeia.

References

• 1 The state Pharmacopoeia Commission of the P RC. Pharmacopoeia of the People’s Republic of China. Beijing; Chemical Industry Press 2000
• 2 Li M F, Xu G J, Xu L S, Jin R L. Survey and identification of commercial example of Shihu (Dendrobium) (I).  Chin Trad Herbal Drugs. 1986;  17 33-7
• 3 Li M F, Xu G J, Xu L S, Jin R L. Survey and identification of commercial example of Shihu (Dendrobium) (II).  Chin Trad Herbal Drugs. 1991;  22 173-8
• 4 Ma G X, Xu G J, Xu L S, Li M F. Survey and identification of commercial example of Shihu (Dendrobium) (III).  Chin Trad Herbal Drugs. 1995;  26 370-2
• 5 Cheung K S, Kwan H S, But P PH, Shaw P C. Pharmacognostical identification of American and oriental ginseng roots by genomic fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR).  J Ethnopharmacol. 1994;  42 67-9
• 6 Shaw P C, But P PH. Authentication of Panax species and their adulterant by random-primed polymerase chain reaction.  Planta Med. 1995;  61 466-9
• 7 Fushimi H, Komatsu K, Isobe M. A new approach for the identification of a Chinese traditional medicine ”Chuanxiong” by 18S ribosomal RNA gene sequences.  Phytomedicine. 1996;  3 387
• 8 Wang Y Q, Zhou K Y, Xu L S, Xu G J. Authentication of the Chinese crude drug ”Wushaoshe” (Zaocys dhumnades) and its substitutes by DNA sequence analysis.  Acta Pharm Sin. 1999;  34 67-71
• 9 Hosokawa K, Minami M, Kawahara K, Nakamura I, Shibata T. Discrimination among three species of medicinal Scutellaria plants using RAPD markers.  Planta Med. 2000;  66 270-2
• 10 Mizukami H, Okabe Y, Kohda H, Hiraoka N. Identification of the crude drug Atractylodes rhizome (Byaku-jutsu) and Atractylodes lancea rhizome (So-jutsu) using chloroplast trnK sequence as a molecular marker.  Biol Pharm Bull. 2000;  23 589-94
• 11 Um J Y, Chung H S, Kim M S, Na H J, Kwon H J, Kim J J. et al . Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP.  Biol Pharm Bull. 2001;  24 872-5
• 12 Yue-Qin C, Ning W, Hui Z, Liang-Hu Q. Differentiation of medicinal Cordyceps species by rDNA ITS sequence analysis.  Planta Med. 2002;  68 635-9
• 13 Hon C C, Chow Y C, Zeng F Y, Leung F C. Genetic authentication of ginseng and other traditional Chinese medicine.  Acta Pharmacol Sin. 2003;  4 841-6
• 14 Cui X M, Lo C K, Yip K L, Dong T T, Tsim K W. Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis.  Planta Med. 2003;  69 584-6
• 15 Wong K L, Wang J, But P P, Shaw P C. Application of cytochrome b DNA sequences for the authentication of endangered snake species.  Forensic Sci Int. 2004;  139 49-55
• 16 Long E O, Dawid B. Repeated genes in eukaryotes.  Annu Rev Biochem. 1980;  49 726-7
• 17 Zhao Z L, Zhou K Y, Dong H, Xu L S. Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.  Planta Med. 2001;  67 381-3
• 18 Wang J B, Zheng W J, Chen J K. Application of ITS sequences of nuclear rDNA in phylogenetic and evolutionary studies of angiosperms.  Acta Phytotaxonom Sin. 1999;  37 407-16
• 19 Tai-Wai Lau D, Shaw P C. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.  Planta Med. 2001;  67 456-60
• 20 Ding X, Xu L S, Wang Z T, Zhou K Y, Xu H, Wang Y Q. Authentication of stems of Dendrobium officinale by rDNA ITS region sequences.  Planta Med. 2002;  68 191-2