SNP identification of Alisma orientale, Alisma plantago-aquatica and Alismatis Rhizoma in the market based on DNA barcoding / 中国药学杂志

OBJECTIVE: To establish a method for the rapid identification of Alismatis Rhizoma based on single nucleotide polymorphism (SNP) molecular marker. METHODS: Total genomic DNA was extracted using improved DNA extraction kit, and the internal tran scribed spacer 2 (ITS2) regions were amplified and annotated by using the hidden Markov model (HMM). In addition, the ITS2 sequences of Alisma orientale and Alisma plantago-aquatica were aligned through Clustal-W, and then SNP was detected to identify Alismatis Rhizoma. RESULTS: The length of A. orientale and A. plantago-aquatica ITS2 sequences was 311 bp. No intra-specific variation was found among the samples of two species, respectively. One stable SNP (A/T) was detected at 165 bp in ITS2 region which was useful for identification of the two species. The result was confirmed by NJ tree method. Furthermore, authentication of commercial Alismatis Rhizoma by SNP molecular marker and NJ tree method indicated that only two medicinal samples (7%) were A. orientale and the others (93%) were A. plantago-aquatica which was not recorded in the Chinese Pharmacopoeia. CONCLUSION: SNP molecular marker can stably and accurately distinguish Alismatis Rhizoma in the market and can offer scientific basis for protection of germplasm resources and cultivation of Alisma.

Molecular identification of aucklandiae radix, vladimiriae radix, inulae radix, aristolochiae radix and kadsurae radix using ITS2 barcode / 中国中药杂志

In order to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix using ITS2 barcodes, genomic DNA from sixty samples was extracted and the ITS2 (internal transcribed spacer) regions were amplified and sequenced. The genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model and the neighbor-joining (NJ) phylogenetic tree was constructed. The results indicated that for Aucklandiae Radix (Aucklandia lappa), Vladimiriae Radix (Vladimiria souliei and V. souliei var. cinerea), Inulae Radix (Inula helenium), Aristolochiae Radix (Aristolochia debilis) and Kadsurae Radix (Kadsura longipedunculata), the intra-specific variation was smaller than inter-specific one. There are 162 variable sites among 272 bp after alignment of all ITS2 sequence haplotypes. For each species, the intra-specific genetic distances were also smaller than inter-specific one. Furthermore, the NJ tree strongly supported that Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix can be differentiated. At the same time, V. souliei (Dolomiaea souliei) and V. souliei var. cinerea( D. souliei var. cinerea) belonging to Vladimiriae Radix were clearly identified. In conclusion, ITS2 barcode could be used to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix. Our study may provide a scientific foundation for clinical safe use of the traditional Chinese medicines.

Identification of atractylodis macrocephalae rhizoma and atractylodis rhizoma from their adulterants using DNA barcoding / 中国中药杂志

Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma were widely used in strengthening spleen under different disease conditions, and were easily and often misused each other. Therefore, DNA barcode was used to distinguish Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma from their adulterants to ensure the safe use. The sequence lengths of ITS2 of Atractylodes macrocephala, Atractylodis Rhizoma (A. lancea, A. japonica and A. coreana) were both 229 bp. Among the ITS2 sequences of A. macrocephala, only one G/C transversion was detected at site 98, and the average GC content was 69.42%. No variable site was detected in the ITS2 sequences of A. lancea. The maximum K2P intraspecific genetic distances of both A. japonica and A. coreana were 0.013. The maximum K2P intraspecific genetic distances of A. macrocephala, A. lancea, A. japonica and A. coreana were less than the minimum interspecific genetic distance of adulterants. The ITS2 sequences in each of these polytypic species were separated into pairs of divergent clusters in the NJ tree. DNA barcoding could be used as a fast and accurate identification method to distinguish Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, from their adulterants to ensure its safe use.

Stability and accuracy of the identification of Notopterygii Rhizoma et Radix using the ITS/ITS2 barcodes / 药学学报

In this study, Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences. Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603-604 bp, while the lengths of ITS2 regions were 228 bp. The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.