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Integrated Analysis for Identifying Radix Astragali and Its Adulterants Based on DNA Barcoding

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Integrated Analysis for Identifying Radix Astragali and Its Adulterants Based on DNA Barcoding Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 843923, 11 pages http://dx.doi.org/10.1155/2014/843923 Research Article Integrated Analysis for Identifying Radix Astragali and Its Adulterants Based on DNA Barcoding Sihao Zheng,1 Dewang Liu,2 Weiguang Ren,1 Juan Fu,1 Linfang Huang,1 and Shilin Chen3 1 InstituteofMedicinalPlantDevelopment,ChineseAcademyofMedical Sciences, Peking Union Medical College, Beijing 100193, China 2 School of Pharmacy, Inner Mongolia Medical University, Inner Mongolia 010080, China 3 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China Correspondence should be addressed to Linfang Huang; [email protected] Received 15 June 2014; Accepted 22 July 2014; Published 27 August 2014 Academic Editor: Robert Henry Copyright © 2014 Sihao Zheng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties. However, methods are needed to help distinguish Radix Astragali from its varied adulterants. DNA barcoding is a widely applicable molecular method used to identify medicinal plants. Yet, its use has been hampered by genetic distance, base variation, and limitations of the bio-NJ tree. Herein, we report the validation of an integrated analysis method for plant species identification using DNA barcoding that focuses on genetic distance, identification efficiency, inter- and intraspecific variation, and barcoding gap. We collected 478 sequences from six candidate DNA barcodes (ITS2, ITS, psbA-trnH, rbcL, matK, and COI) from 29 species of Radix Astragali and adulterants. The internal transcribed spacer (ITS) sequence was demonstrated as the optimal barcode for identifying Radix Astragali and its adulterants. This new analysis method is helpful in identifying Radix Astragali and expedites the utilization and data mining of DNA barcoding. 1. Introduction Several PCR-based molecular methods have been developed, providing an alternative means of identification. Multiplex Radix Astragali (Huang Qi), a commonly used Chinese PCR methods of DNA fragment analysis, such as randomly medicinal material, is mainly sourced from the plants amplified polymorphic DNA (RAPD) [12] or amplified frag- of Astragalus membranaceus and Astragalus mongholicus ment length polymorphism (AFLP) [13],areunstableforthe according to Chinese Pharmacopoeia (2010 edition). Radix results to identify. DNA barcoding is a widely used molecular Astragali is widely used for its antiperspirant, antidiuretic, marker technology, first proposed by Hebert et al. [14, 15]. and antidiabetic properties and as a tonic drug [1–3]. It It uses a standardized and conserved, but diverse, DNA possesses various beneficial compounds, including astraga- sequence to identify species and uncover biological diversity losides, isoflavonoids, isoflavones, isoflavan, and pterocarpan [16, 17]. In previous studies, various coding sequences for glycosides [4–6]. identifying Radix Astragali and its adulterants have been Due to the high market demand for Radix Astragali, used, such as the 5S-rRNA spacer domain [18], 3 untrans- a diverse group of adulterants with similar-morphological lated region (3 UTR) [19], ITS (internal transcribed spacer characteristics from genuses, such as Astragalus, Hedysarum, region) and 18S rRNA [3, 20, 21], ITS2 [22], ITS1 [6], matK and Malva areoftenusedinitsstead[7]. The traditional (maturase K) and rbcL (ribulose 1, 5-bisphosphate carboxy- methods used to identify Radix Astragali for use as a lase) of chloroplast genome, and coxI (cytochrome c oxidase medicinal material, such as morphological and microscopic 1) of the mitochondrial genome [23]. However, sequence identification [8], thin-layer chromatography and Ultraviolet analysis was mainly focused on genetic distance, variable spectroscopy [9], Fourier Transform infrared spectroscopy sites, amplified polymorphisms, and the use of a modified (FTIR) [10], and high performance liquid chromatography neighbor-joining (NJ) algorithm, Bio-NJ tree, which were (HPLC) [11], all, require specialized equipment and training. basic analyses limited to particular species. A more effective 2 Evidence-Based Complementary and Alternative Medicine Table 1: Taxon sampling information of astragalus and its adulterants. Experiment number species Sampling spot S1-S5 Astragalus membranaceus Shaanxi China SD1-SD9 Astragalus membranaceus Shaanxi China GS1-GS6 Astragalus mongholicus Gansu China NM1-NM10 Astragalus mongholicus Neimeng China SX1-SX10 Astragalus mongholicus Shanxi China HHQ1-HHQ7 Astragalus chinensis Beijing China CY1-CY6 Astragalus scaberrimus Beijing China JK1-JK3 Malva pusilla Shaanxi China MX Medicago sativa Shaanxi China HH1-HH7 Melilotus officinalis Shaanxi China HQ1-HQ12 Hedysarum polybotrys Gansu China XJ Astragalus adsurgens Beijing China method of molecular identification is necessary. The current Genomic DNA Kit, Tiangen Biotech Co., China). We made study evaluates the identification reliability and efficiency of the following modifications to the protocol: chloroform was DNAbarcodingfortheidentificationofRadixAstragaliusing diluted with isoamyl alcohol (24 : 1 in the same volume) and six indicators of genetic distance, identification efficiency, buffer solution GP2 with isopropanol (same volume). The ∘ intra- and interspecific variation, gap rate, and barcoding gap. powder, 700 Lof65CGP1,and1L -mercaptoethanol Six barcodes were selected for identification because they were mixed for 10–20 s before being incubated for 60 minutes ∘ are commonly used in plant, especially in medicinal plant. at 65 C. Then, 700 L of the chloroform:isoamyl alcohol We collected Radix Astragaliand several of its adulterants mixture was added and the solution was centrifuged for 5 reported in previous research and downloaded the genetic minutes at 12000 rpm (∼13400 ×g). Supernatant was removed sequences from the GenBank database. A total of 29 species and placed into a new tube before adding 700 Lisopropanol (including 19 species of Astragalus) and 478 sequences from and blending for 15–20 minutes. The mixture was centrifuged six barcodes were used to validate the new method for in CB3 spin columns for 40 s at 12000 rpm. The filtrate identifying Radix Astragali and adulterants and to accelerate was discarded and 500 L GD (adding quantitative anhy- the data utilization of DNA barcoding. drous ethanol before use) was added before centrifuging at 12000 rpm for 40 s. The filtrate was discarded and 700 L 2. Materials and Methods PW (adding quantitative anhydrous ethanol before use) was used to wash the membrane before centrifuging for 40 s at 2.1. Materials Information. A total of 77 specimens were 12000 rpm. This step was repeated with 500 LPW,followed collected from two origins of Radix Astragali, along with by a final centrifuge for 2 minutes at 12000 rpm to remove seven adulterants. Radix Astragali specimens were collected residual wash buffer. The spin column was dried at room fromInnerMongolia,Shaanxi,andGansuprovincesinthe temperature for 3–5 minutes and then centrifuged for 2 People’s Republic of China, which are the main producing minutesat12000rpmtoobtainthetotalDNA. areas. The collection information is shown in Table 1.All General PCR reaction conditions and universal DNA corresponding voucher specimens were deposited in the barcode primers were used for the ITS, ITS2, and psbA-trnH Herbarium of the Institute of Medicinal Plant Development at barcodes, as presented in Table 2 [24–26]. PCR amplification the Chinese Academy of Medical Sciences in Beijing, China. was performed on 25-L reaction mixtures containing 2 L The GenBank accession number of the ITS2 in this experi- DNA template (20–100 ng), 8.5 L ddH2O, 12.5 L2× Taq ment was orderly KJ999296–KJ999344, the accession num- PCR Master Mix (Beijing TransGen Biotech Co., China), and ber of ITS sequences was orderly KJ999345–KJ999416, and 1/1-L forward/reverse (F/R) primers (2.5 M). The reaction the accession number of psbA-trnH was orderly KJ999256– mixtures were amplified in a 9700 GeneAmp PCR system KJ999295. The sequences added in the subsequent analysis, (Applied Biosystems, USA). Amplicons were visualized by including ITS, ITS2, psbA-trnH, matK, and rbcL, were down- electrophoresis on 1% agarose gels. Purified PCR products loaded from the GenBank database. were sequenced in both directions using the ABI 3730XL sequencer (Applied Biosystems, USA). 2.2. DNA Extraction, PCR Amplification, and Sequencing. The material specimens were naturally dried and 30mg 2.3. Sequence Assembly, Alignment, and Analysis. Sequencing of dried plant material was used for the DNA extraction. peak diagrams were obtained and proofread, and then contigs Samples were rubbed for two minutes at a frequency of were assembled using a CodonCode Aligner 5.0.1 (Codon- 30 r/s in a FastPrep bead mill (Retsch MM400, Germany), Code Co., USA). Complete ITS2 sequences were obtained and total genomic DNA was isolated from the crushed using the HMMer annotation method, based on the Hidden material according to the manufacturer’s instructions (Plant Markov model (HMM) [27]. All of the sequences were Evidence-Based Complementary and Alternative Medicine 3 Table2:PrimersandPCRreactionconditions. Primer name Primer sequences
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