A set of novel microsatellite markers developed for the traditional Tibetan medicinal plant Halenia elliptica (Gentianaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed in the traditional Tibetan medicinal plant Halenia elliptica D. Don to investigate its genetic diversity and population genetic structure. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 24 primer sets were identified in two wild populations. Of these primers, 12 displayed polymorphisms and 12 were monomorphic. The number of alleles per locus ranged from 2 to 6, with a mean of 3.9. The expected (H(E)) and observed (H(O)) heterozygosities ranged from 0.191 to 0.784 and from 0.417 to 0.917, respectively. All these primers successfully amplified in two close relatives of H. elliptica, Swertia bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C. B. Clarke and S. tetraptera Maxim. CONCLUSIONS: These markers will facilitate further studies on the population genetics of Halenia elliptica and its allied species.

Application of LightCycler polymerase chain reaction and melting curve analysis to the authentication of the traditional Chinese medicinal plant Cimicifuga foetida.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and fluorescence melting curve analysis of LightCycler real-time polymerase chain reaction products were exploited for their applications in the authentication of the traditional Chinese medicinal plant Cimicifuga foetida from four substitutes: C. heracleifolia, C. dahurica, C. acerina, and C. simplex. According to the melting temperature--which is a function of the GC/AT ratio, length, and nucleotide sequences of the amplified product--C. foetida was differentiated from C. heracleifolia, C. dahurica, C. acerina, and C. simplex. Melting curve analysis offers a rapid and reliable method for the authentication of the traditional Chinese medicinal plant C. foetida.

Identification and quantification of the traditional Chinese medicinal plant Gentiana macrophylla using Taqman real-time PCR.

Gentiana macrophylla Pall. is a commonly used antirheumatic herb. There are four species of Gentiana recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as G. macrophylla, and thus the therapeutic effects of G. macrophylla are not achieved. A novel one-step methodology based on real-time polymerase chain reaction (PCR) technology has been developed for the identification of G. macrophylla. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.

Application of real-time scorpion PCR for authentication and quantification of the traditional Chinese medicinal plant Drynaria fortunei.

DNA sequence analysis of the trnl-trnF spacer region and real-time scorpion polymerase chain reaction were exploited for their applications in the differentiation and quantification of the traditional Chinese medicinal plants Drynaria fortunei from five related adulterants: D. mollis, D. quercifolia, D. rigidula, D. sparsisora, and Pseudodrynaria coronans. The data demonstrated that variations in the trnl-trnF spacer regions were very low at the intraspecific level but extremely high at the interspecific level, meaning that they could be easily distinguished at the DNA level. The sequence difference allowed effective and reliable differentiation of D. fortunei from adulterants by real-time scorpion PCR. Furthermore, a quantification methodology was carried out to quantify D. fortunei.

Differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from adulterants by TaqMan real-time polymerase chain reaction.

DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional chinese medicinal plants euphorbia humifusa and e. maculata from three related adulterants e. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii.

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA RPL16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA RPL16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.