Orchidaceae, commonly referred as the orchid family, is the second largest family of flowering plants, comprising more than 25,000 species distributed in 880 genera (Gutierrez, 2010). Dendrobium is one of the largest genera in Orchidaceae and includes ≈1600 species (Bechtel et al., 1992). Seventy-four Dendrobium species and two variations are found in China, and at least 12 species have been identified in Taiwan (Su, 2000). The processed dried stems of certain Dendrobium species are known as “Shi-hu” [herbal dendrobii (stems of Dendrobium)] that contain compounds with biological activities. Therefore, Shi-hu has been used for medical treatment and as a functional health food in many Asian countries for centuries.
According to the Pharmacopoeia of the People’s Republic of China (China Medical Science and Technology Press, 2010), four species, D. catenatum, D. nobile, D. chrysotoxum, and D. fimbriatum, are used as the authorized sources for herbal dendrobii. However, other Dendrobium species are also used as clinical substitutes: in the Taiwan herb market, D. tosaense, D. linawianum, D. moniliforme, D. hercoglossum, and D. nobile are common medicinal plants used as the sources for Shi-hu. The former three species are found distributed in the mountain areas of Taiwan, whereas D. hercoglossum and D. nobile are mainly imported from China (Hsieh, 2004). D. tosaense has drawn more attention as an important herbal dendrobii in Taiwan as a result of its natural habitat distribution (Tang and Cheng, 1984) and fairly good growth rate in comparison with other Dendrobium species. The mass production of D. tosaense by tissue culture has also been well established (Lo et al., 2004b). Furthermore, the plant produces a major bioflavonoid, quercetin, which possesses antioxidant (Gutierrez, 2010; Lo et al., 2004a), antihypertensive, anti-inflammatory, and anticarcinogen properties (Middleton and Kandaswami, 1993), making D. tosaense a good source of medicinal Dendrobium.
A major problem in Asian medicinal herb markets is the use of imitations and adulterants. Adulterants that are composed of cheaper and common orchids are found on the market (Ma et al., 1995), and the similarity in appearance with the authentic counterparts makes Shi-hu difficult to discriminate. Therefore, the authentication of medicinal Dendrobium species is important to ensure the therapeutic quality and safety of this Shi-hu. Many attempts have been made to identify Dendrobium species. In general, the differentiation relies on the morphology and anatomy of the fresh materials (Carlsward et al., 1997); however, this approach requires an empirical taxonomist for reliable identification. Pharmacognostic and chemical analyses may be used in the identification of medicinal Dendrobium. However, these analytical approaches are not always applied for species identification. In contrast, a molecular genetics approach offers a reliable and effective means of Dendrobium species identification. To date, several molecular analytical strategies have been applied to identify Dendrobium species. A suppression subtraction hybridization array was used to identify five Dendrobium species (Li et al., 2005). The internal transcribed spacer region in the rDNAs encoding nuclear rRNA genes and the plastid and mitochondrial genomes were used as DNA bar codes for the authentication of plant species (Tsai et al., 2004; Yao et al., 2009). Amplified fragment length polymorphism technology was also used to screen for genetic markers within a Dendrobium hybrid that was associated with a phenotype of interest (Hong et al., 2000). Random amplified polymorphic DNA was used to identify the genetic diversity of D. officinale (Ding et al., 2009) and to construct linkage maps of D. officinale and D. hercoglossum (Xue et al., 2010). In addition, adapter ligation-mediated allele-specific amplification was applied to authenticate three Dendrobium species, including D. aphyllum, D. devonianum, and D. officinale (Zhang et al., 2009). A set of polymorphic markers from 10 microsatellite loci were used to analyze the genetic diversity of D. fimbriatum (Fan et al., 2009). A species-specific inter-simple sequence repeat analysis was also used to authenticate several populations of D. officinale (Shen et al., 2006) and to evaluate the genetic diversity of 31 Dendrobium species (Wang et al., 2009).
The ITS region (ITS1-5.8S-ITS2) is highly repeated, up to many thousands of copies, in plant nuclear genomes (Baldwin et al., 1995), and this high copy number and its location between the conserved sequences of 18S and 26S RNA coding regions promote the detection, amplification, cloning, and sequencing of rDNA. Because the ITS1 and ITS2 regions exhibit high divergence among species and populations, they are suitable targets for species identification; in contrast, 18S, 5.8S, and 26S exhibit lower divergence among species and have been explored to study more distant relationships at the order or family level (Yip et al., 2007). The rapid concerted evolution of the ITS region through unequal crossing-over and gene conversion promotes the accurate reconstruction of species relationships according to the ITS sequences (Baldwin et al., 1995; Hillis et al., 1991).
Based on the ITS sequences, the multiplex PCR method can be used to amplify target-containing samples by mixing specific primers in a single PCR. This approach has been widely used to identify microbes and medicinal plants (Jigden et al., 2010). The amplification refractory mutation system, which has also been described as allele-specific PCR, amplifies on the basis of single nucleotide polymorphisms (SNPs) of different sequences (Newton et al., 1989) and has been successfully used to authenticate orchids, including D. officinale (Ding et al., 2003) and D. loddigesii (Qian et al., 2008).
The aims of this study are as follows: 1) to establish a database of the ITS region of 20 Dendrobium species, because such data will provide information to identify Dendrobium species; 2) to determine the evolutionary relationships of these 20 Dendrobium species; and 3) to discriminate six of the herbal dendrobii from the other 14 species using multiplex PCR. Among these six species, five are commonly used herbal materials for Shi-hu in Taiwan, including D. tosaense, D. nobile, D. linawianum, D. hercoglossum, and D. moniliforme; whereas D. huoshanense, although seldom found on the market as a result of its slow growth rate and small size after processing, is an authorized herbal plant. Another aim of the study was to specifically identify D. tosaense using ARMS analysis. The establishment of an ITS database and the PCR-based methods will assist and promote effective and accurate identification of Dendrobium species.
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