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Molecular Authentication of Magnoliae Flos Using Robust SNP Marker Base on trnL-F and ndhF Region
Molecular Authentication of Magnoliae Flos Using Robust SNP Marker Base on trnL-F and ndhF Region
Korean Journal of Plant Resources. 2015. Jun, 28(3): 341-349
Copyright © 2015, The Plant Resources Society of Korea
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : April 04, 2015
  • Accepted : June 06, 2015
  • Published : June 30, 2015
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About the Authors
Min-Kyeoung Kim
KM Fundamental Research Division, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea
Jong-Hun Noh
Korean Ginseng Center for Most Valuable Products & Ginseng Genetic Resource Bank, Kyung Hee University, Yongin 446-701, Korea
Deok-Chun Yan
Korean Ginseng Center for Most Valuable Products & Ginseng Genetic Resource Bank, Kyung Hee University, Yongin 446-701, Korea
Sanghun Lee
KM Fundamental Research Division, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea
Hee-Nyeong Lee
Korean Ginseng Center for Most Valuable Products & Ginseng Genetic Resource Bank, Kyung Hee University, Yongin 446-701, Korea
Chi-Gyu Jin
Korean Ginseng Center for Most Valuable Products & Ginseng Genetic Resource Bank, Kyung Hee University, Yongin 446-701, Korea
jinck@khu.ac.kr
Abstract
Magnoliae Flos (Sini in Korean) is one of the most important oriental medicinal plants. In the Korean Herbal Pharmacopeia, the bud of the all species in Manolia denudate and Manolia genus were regarded as the botanical sources for ‘Sini’. Most the dried bud of Manolia denudata , Manolia biondii and Manolia sprengeri were used as ‘Xin-yi’ in China. Therefore, the purpose of this study was to determine and compare the ‘ Magnolia ’ species, four species including Manolia denudata , M. biondii , M. liliiflora and M. Kobus were analysis of sequencing data revealed DNA polymorphisms. The based on tRNA coding leucine/phenylalanine ( trn L-F) and NADH-plastoquinone oxidoreductase subunit 5 ( ndh F) sequences in chloroplast DNA. For the identification of ‘ Magnolia ’ species, polymerase chain reaction (PCR) analysis of chloroplast DNA regions such as ndh F have proven an appropriate method. A single nucleotide polymorphism (SNP) has been identified between genuine “Sini” and their fraudulent and misuse. Specific PCR primers were designed from this polymorphic site within the sequence data, and were used to detect true plants via multiplex PCR.
Keywords
Introduction
Magnoliae Flos (Sini in Korean), the dried bud of Manolia denudate including Manolia genus is one of the most widely used herbal medicinal plants in Korean Herbal Pharmacopeia (KHP, 4 th ). Among the “ Magnolia ” species, four species, Manolia denudata , M. biondii , M. liliiflora and M. kobus were most used as publicly certified medicinal materials in Korean Herbal Pharmacopeia (KHP, 5 th ).
Magnoliae Flos has an astringent effect, which can improve local blood circulation and promote the absorption of secretions, thereby diminishing inflammation, clearing nasal passage, and relieving or eliminating symptoms ( Wang ., 1996 ), it also has an anti-allergic effect, which can effectively fight against the nasal itching, sneezing, runny nose, and other symptoms caused by allergic rhinitis ( Kim ., 2002 ). However, it is the absence of the test component for each of the four species. Also, the dried bud of four species are always misidentified as Magnoliae Flos due to their morphological similarities, most of medicinal plant products in the markets are packaged of powders of slices and Processed things that no longer bear the original morphological features of the plants ( Park ., 2006 ; Jigden ., 2010 ; Jin ., 2013 ).
Traditional authentication methods, which have relied on morphological and histological differences, are limited and quite often unreliable. Chemical analysis are significantly affected by environmental growth conditions as well as storage conditions that in comparison, DNA analysis by molecular techniques is not influenced by growth stage and environmental conditions of plants ( Zhu ., 2004 ).
Several molecular methods, such as arbitrarily primed polymerase chain reaction (AP-PCR) ( Cheung ., 1994 ), random amplified polymorphic DNA (RAPD) ( Cui ., 2003 ; Shim ., 2003 ; Shaw and But, 1995 ), loop-mediated isothermal amplification (LAMP) ( Sasaki ., 2008 ), sequence characterized-amplified region (SCAR) ( Choi ., 2008 ; Wang ., 2001 ), restriction fragment length polymorphism (RFLP) ( Ngan ., 1999 ), amplification fragment length polymorphism (AFLP) ( Ha ., 2002 ), and DNA microarray ( Zhu ., 2008 ) have previously been described. The main drawback of RAPD are lack of reliability and reproducibility. RFLP and AFLP need stringent reaction conditions and tedious operation, and they are not suitable for identification of processed sample due to the degradation of genomic DNA.
In this study, we describe general ways to differentiate medicinally important plants in a more reproducible and robust approach by analyzing Single nucleotide polymorphisms (SNP). For the SNP analysis, used regions include ribosomal genes, chloroplast genes and mitochondrial genes.
In this case, in order to determine the base sequence of trn L (tRNA-Leu) and ndh F intron region were targeted for molecular analysis and these regions were proved to be useful for discrimination of M. denudata , M. biondii , M. liliiflora and M. kobus .
Chloroplast DNA ( cp DNA) intron and intergenic spacer sequence is used for evolutionary and phylogenetic study ( Olmstead and Palmer 1994 ). The cpDNA trn L (tRNA-Leu) and ndh F intron region in land plants comprising the trn L (UAA) intron and trn L (UAA)- trn F (GAA) noncoding intergenic spacer is one of most widely used chloroplast markers for phylogenetic analysis in plants ( Quandt and Stech, 2004 ). It has been used successfully to infer phylogenetic relationships within and among angiosperm families ( Olmstead ., 1992 )
It was possible to test paternity of medicinal herbs, as it is possible to identify true Bos taurus coreanae and young antlers of deers, using genetic base sequencing ( An ., 2006 ). To identify plants using DNA base sequencing, chloroplast DNA or trn L-F (tRNA-Leu/Phe) / ndh F (NADH dehydrogenase subunit F) is frequently used as it is found only in plants ( Lang ., 2006 ).
A marker that recognizes four species of the genus Magnolia ( Manolia denudata , M. biondii , M. liliiflora and M. kobus ) for herbal use was developed after determining the trn L-F region and base sequencing ndh F gene and verifying its SNP.
Although traditional herbal medicine industry enrooted in medicinal plants as resources is one of the major medical organizations partnering with Western medicine, it currently imports more than 90% of raw materials from China, and there are cases of adverse reactions because of mixture of unsorted ingredients, improper applications and misunderstandings due to lack of manufacture and distribution management system. Therefore, the aim of this research is to trace the origin of Magnoliae Flos through analysis of DNA base sequence, and to develop identifying technology such as SNP markers so that there will be standardization of distribution of the herbal Magnoliae Flos.
This research promotes to verify authenticity of Magnoliae Flos and to develop identification methods, as demanded by Korea Food and Drug Administration-linked Center for Herbal Medicine.
Materials and Methods
- Plant
The collected samples of Magnoliae Flos are shown in Table 1 . Ten samples of herbal Magnoliae Flos currently distributed in Korea and China were purchased. And the standard ten samples were collected from the following places, The Ok-cheon medicinal plants authentication center of Korea Food and Drug Administration (http://agri.oc.go.kr), Kyung Hee University in Su-won, Po-cheon, and Young-cheon. Other eight samples were collected from substance separation and analysis team at Andong National University. All samples were correctly identified by the sequence of trn L-F and ndh F of NCBI-registered Magnolia plants were screened and confirmed.
List of ‘Magnoliae Flos (Sini)’ samples used in this study
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List of ‘Magnoliae Flos (Sini)’ samples used in this study
- DNA extraction
A total of twenty eight collected samples were frozen using liquid nitrogen, ground using porcelain mortar and pestle, and the genomic DNA were extracted using G-spin TM Genomic DNA Extraction Kit (iNtRON, Seongnam, Koera) and each dried sample was isolated using a modified cetyltrimethylammonium bromid (CTAB) method ( Murray ., 1980 ).
- PCR amplification of thetrnL (tRNA-Leu) andndhF (ndhF) intron region
In order to determine the base sequence of trn L-F, universal primers trn LF-c (forward) and trn LF-f (reverse) of the trn L-F region were used in PCR amplification. The base sequence of the primers were trn LF-c (5'-CGA AAT CGG TAG ACG CTA-3') and trn LF-f (5'-ATT TGA ACT GGT GAC ACG AG-3') ( Taberlet ., 1991 ).
The conditions of running PCR were set as the following: pre-denaturation at 96℃ for 2 minutes; denaturation at 96℃ for 30 seconds; annealing at 57℃ for 30 seconds; and extension at 72℃ for 2 minutes, for 36 cycles. Universal primers ndh F-F1 (forward) and ndh F-R1 (reverse) of the ndh F region were used in PCR amplification to determine the base sequence of ndh F. The base sequence of the primers were ndh F-F1 (5'-TTG GGA ATT GGT GGG AAT GTG-3') and ndh F-R1 (5'-TTC CTA TGG ACC CAA CGA AC-3') ( Zhang ., 2003 ), and the conditions of running PCR were set as the following: pre-denaturation at 95℃ for 3 minutes; denaturation at 95℃ for 30 seconds; annealing at 61℃ for 30 seconds; and extension at 72℃ for 2 minutes, for 36 cycles. Each PCR amplification was performed in a volume of 20㎕, and the reaction mixture consisted of each of the primers at a concentration of 0.5 ㎛, 20 ng of template DNA. The PCR products were migrated on a 1.0% agarose gel electrophoresis and detected by ethidium bromide staining under UV.
- Sequencing and DNA sequence analysis
The PCR products were purified using a GENEALL PCR SV Purification Kit (GeneAll Biotechnology, Seoul, Koera) per the manufacturer’s instructions and then sequenced by Genotech, Inc (Genotech, Daejeon, Korea). The DNA sequence of the trn L (tRNA-Leu) and ndh F ( ndh F) intron regions obtained in sequencing experiments were compiled using SeqMan software, and the sequence were edited with BioEdit program ( Hall ., 1999 ). Multiplex sequence alignments were performed using online Clustal W2 program (http://www.ebi.ac.uk/Tools/clustalw2/).
- Design of specific primers
Specific primer were designed for M. denudata , M. liliiflora , M. Kobus and M. biondii , respectively, on the basis of the DNA polymorphisms detected ( Table 2 ). In order to design specific primers to recognize the four species of Magnolia ( M. denudata , M. liliiflora , M. kobus , M. biondii ) through DNA obtained from plants and herbal Magnoliae Flos used in this research, SNP unique to ndh F of each species were determined.
Specific primer design adjacent to SNP position using the mismatching technology
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Specific primer design adjacent to SNP position using the mismatching technology
There was no SNP unique to M. kobus , and therefore “SIkbnF” specific primer was designed to recognize M. kobus and M. biondii , a specific primer “SIbinF” was designed only to identify M. biondii . Likewise, no unique SNP could be verified in M. denudata that after designing a specific primer “SIdlnF” to recognize M. denudata and M. liliiflora , a specific primer “SIlinF” was designed only to identify M. liliiflora . Specific primers were designed through “mismatching” technology ( Kim ., 2005 ; Shiokai ., 2008 ; Hayashi ., 2004 ).
- Multiplex PCR
Molecular authentication of M. denudata , M. liliiflora , M. Kobus and M. biondii was performed using multiplex-PCR. Six primers ( ndh F-F1, SIbinF, SIkbnF, SIdlnF, SIlinF and ndh F-R1) were used simultaneously in multiplex PCR amplification. The reaction mixture was identical to the one described earlier except the concentrations of ndh F-F1, SIkbnF, SIdlnF, SIlinF and ndh F-R1 were 0.63 ㎛, 0.31 ㎛, 0.63 ㎛, 1.13 ㎛, 0.5 ㎛ and 0.81 ㎛ respectively. PCR amplification was performed in a total volume of 20㎕. The conditions for DNA amplification were set as the following: pre-denaturation at 95℃ for 3 minutes; denaturation at 95℃ for 30 seconds; annealing at 61℃ for 30 seconds; and extension at 72℃ for 2 minutes, for 33 cycles. The PCR products were visualized on a 1% agarose gel.
- Results and Discussion
Chloroplast DNA ( cp DNA) sequence variations are now widely used to investigate interspecific relationships among angiosperms and other plants ( palmer ., 1988 ).
In this study, each cp DNA was separated and the regions of trn L-F and ndh F were amplified using PCR, in order to verify the information on the base sequence of the collected 28 samples of Magnoliae Flos. As a result, each sample yielded 950 bp and 1,248 bp of trn L-F band and ndh F band, respectively ( Fig. 1 , Fig. 2 ). As a result of analysis of base sequencing of trn L-F region, the samples were divided into three groups by the different base of SNP position (333 bp and 835 bp).
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Amplification of four species ‘Magnoliae Flos (Sini)’ DNA sequence (trnL-F).
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Amplification of four species ‘Magnoliae Flos (Sini)’ DNA sequence (ndhF).
Twenty eight plants samples of Magnoliae Flos trn L-F region DNA sequences were collected form NCBI, and aligned in Bioedit program with ClustalX program and a phylogenetic tree was built using Mega4 program. The A group, B group and most group were classified cousin relationship with M. denudate , M. Kobus and M. biondii is classified distant relationship ( Fig. 3 ).
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The Neighbor-joining tree of trnL-F sequences of species belonging to the genus Magnolia.
In contrast, the analysis of base sequencing of ndh F domain divided the samples into four groups by the different base of SNP position (213 bp, 395 bp, 517 bp and 926 bp, respectively) ( Table 3 ).
The base of SNP position and classification of Magnoliae Flos (Sini)
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The base of SNP position and classification of Magnoliae Flos (Sini)
The t RNA coding sequences ( trn L-F), the regions cannot be used for authentication of Magnoliae Flos due to their less polymorphism. In comparison, the ndh F (NADH dehydrogenase subunit F noncoding region was targeted for molecular analysis and differentiation among Magnoliae Flos. These regions can be used for authentication of M. denudata , M. liliiflora , M. Kobus and M. biondii .
The ndh F noncoding regions of four species were PCR-amplified using the ndh F-F1 and ndh F-R1 universal primer set has 1,248 bp of band. All the herbal Magnoliae Flos that are distributed currently in Korea and China are verified as M. biondii . M. denudata and M. liliiflora are related closely in their familial lines, and this could be verified through the analysis of divergence between the Magnolia species. The difference came out to be only 1 bp out of 1,143 bp of the aligned sequence for divergence analysis ( Table 4 ).
Nucleotide divergences ofndhF regions
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Nucleotide divergences of ndhF regions
Twenty eight plants samples of Magnoliae Flos ndh F region DNA sequences were collected form NCBI, and aligned in Bioedit program with ClustalX program and a phylogenetic tree was built using Mega4 program. The A group , B group, C group and most group were classified cousin relationship with M. denudate , M. Kobus , M. liliiflora and M. biondii is classified distant relationship ( Fig. 4 ).
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The Neighbor-joining tree of ndhF sequences of species belonging to the genus Magnolia.
The ndh F universal band at 1,248 bp was used as control in order to verify the presence or absence of PCR amplification. Their sequences were deposited in GeneBank (KP742958-KP74296).
Multiplex alignment of ndh F noncoding region of M. denudata , M. liliiflora , M. Kobus and M. biondii were performed with CLUSTALX program. Specific primer were designed for M. denudata , M. liliiflora , M. Kobus and M. biondii based on the detected SNP sites. Molecular discrimination of Magnoliae Flos was conducted using multiplex PCR with the six primers described. The combination of six specific primers described, as shown in Fig. 5 , yield expected amplicons for different species. The specific primer “SIbinF” developed a specific band at 1,032 bp, which is amplified only in M. biondii ; “SIkbnF” showed a specific band at 860 bp, amplified only in M. kobus / M. biondii ; “SIdlnF” expressed at 738 bp, amplified uniquely in M. denudata / M. liliiflora ; and “SIlinF” band appeared at 328 bp, amplified only in M. liliiflora ( Fig. 5 ).
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Multiplex PCR for identification of four species of Magnoliae Flos (Sini).
The specific primer “SIbinF” developed a specific band at 1,032 bp, which is amplified only in M. biondii ; “SIkbnF” showed a specific band at 860 bp, amplified only in M. kobus / M. biondii ; “SIdlnF” expressed at 738 bp, amplified uniquely in M. denudata / M. liliiflora ; and “SIlinF” band appeared at 328 bp, amplified only in M. liliiflora .
Therefore, authentic Magnoliae Flos can be identified through its specific band from the influx of commercially available herbal plants, distinguished by its SNP markers (specific primers) using multiplex-PCR. The results obtained through this research can be expected not only to verify the origin of Magnoliae Flos that is circulated in today’s market but also to recognize its species, as well as to separate from inauthentic and misused plants.
The described method has important implications in both the production and sale of this medicinal products, allowing for the Prevention of fraud and misuse, and also revealing the possible presence of other with cheaper plant material. This method is reliable, efficient, and can be used for numerous repeated tests of many medicinal plants, and the methodology presented in this study can be adapted for authentication of other medicinal materials.
Acknowledgements
This work was supported by the YS personnel expenditure (490009-1297) from National Research Council of Science & Technology (NST) and the grant K15060 awarded to Korea Institute of Oriental Medicine (KIOM) from Ministry of Education, Science and Technology (MEST), Korea.
References
An S.M. , Ryuk J.A. , Kim Y.H. , Chae B.C. , Kim H.J. , Kim K.H. , Kang K.K. , Ko B.S. , Lee. M.Y. 2006 Genetic analysis of polygonati rhizoma and polygonati odorati rhizoma using random amplified microsatlite polymorphism Korean J Medicinal Crop Sci. 14 125 - 129
Cheung K.S. , Kwan H.S. , But P.P. , Shaw. P.C. 1994 Pharmaceutical identification of American and oriental ginseng roots by genomic fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR) J Ethnopharmacol. 42 67 - 69
Choi Y.E. , Ahn C.H. , Kim B.B. , Yoon E.S. 2008 Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicas C. A. Meyer Biological and Pharmaceutical Bulletin 31 135 - 138
Cui X.M. , Lo C.K. , Yip K.L. , Dong K.W. , Sim K.T. 2003 Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) Analysis Planta Medica 69 584 - 586
Jigden B. , Wang H. , Kim Y.J. , Samdan N. , In J.G. , Yang D.C. 2010 Molecular identification of oriental medicinal plant Schizonepeta tenuifolia bunge (‘Hyung-Gae’) by multiplex PCR Plant Biotechnol Res. 4 223 - 228
Jin C.G. , Kim M.K. , Kim J.Y. , Sun M.S. , Kwon W.S. , Yang D.C. 2013 Molecular authentication of Morus folium using mitochondrial nad7 intron 2 region Korean J. Plant Res. 26 (3) 397 - 402
Ha W.Y. , Shaw P.C. , Liu J. , Yau F.C. , Wang J. 2002 Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD) Journal of Agricultural and Food Chemistry 50 1871 - 1875
Hall T.A. 1999 BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucleic Acids Symp. Ser. 41 95 - 98
Hayashi K. , Hashimoto N. , Daigen M. 2004 Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus Theor. Appl. Genet. 108 (7) 1212 - 1220
Kim H.M. , Yi J.M. , Lim K.S. 2002 Magnoliae Flos inhibits mast cell-dependent immediate-type allergic reactions Pharmacological Res. 32 (2) 107 - 111
Kim M.Y. , Van K. , Lestari P. 2005 SNP identification and SNP marker development for a GmNARK gene controlling supernodulation in soybean Theor. Appl. Genet. 110 (6) 1003 - 1010
Kim M.K. , Baigalmaa J. , Sun H. , Noh J.H. , Kim S.Y. , Yang D.C. 2008 Phylogenetic analysis of Ji-Mo (Anemarrhena asphodeloides) on the basis of chloroplast DNA sequences Korean J Medicinal Crop Sci. 16 (1) 20 - 26
Kim M.K. , Jang G.H. , Yang D.C. , Lee S.H. , Lee H.N. , Jin C.G. 2014 Molecular authentication of Acanthopanacis cortex by multiplex-PCR analysis Tools Korean J. Plant Res. 27 (6) 680 - 686
Lang P. , Dane F. , Kubisiak T.L. 2006 Phylogeny of Castanea (Fagaceae) based on chloroplast trnL-F sequence data Tree Genetics & Genomes 2 132 - 139
Murray M.G , Thompson W.F. 1980 Rapid isolation of high molecular weight plant DNA Nucleic acids Research 8 4321 - 4325
Ngan F. , Shaw P. , But P.P. , Wang J. 1999 Molecular authentication of Panax species Phytochemistry 50 787 - 791
Olmstead R.G. , Scotland R. , Wagstaff S. , Sweere J. , Reeves P. 1992 Application of the chloroplast gene ndhF to angiosperm phylogenetic studies Plant Molecular Evolution Newsletter 2 27 - 30
Olmstead R.G , Palmer J.D. 1994 Chloroplast DNA systematics: a review of methods and data analysis American Journal of Botany 81 1205 - 1224
Palmer J.D. , Jansen R.K. , Michaes H.J. , Chase M.W. , Manhart d J.R. 1988 Chloroplast DNA variation and plant phylogeny Ann Missouri Bot Garden 75 1180 - 1206
Park M.J. , Kim M.K. , In J.G. , Yang D.C. 2006 Molecular identification of Korean ginseng by amplification refractory mutation system-PCR Food Res. Int. 39 568 - 574
Quadt D , Stech M. 2004 Molecular evolution of the trnTUGU trnFGAA region in Bryophtes Plant Biology 6 (5) 545 - 554
Sasaki Y. , Komatsu K. , Nagumo S. 2008 Rapid detection of Panax ginseng by loop-mediated isothermal amplification and its application to authentication of ginseng Biological and Pharmaceutical Bulletin 31 1806 - 1808
Shiokai S. , Kitashiba H. , Shirasawa K. 2008 Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis Molecular Breeding 23 (2) 329 - 336
Shaw P.C , But P.P. 1995 Authentication of Panax species and their adulterants by random-primed polymerase chain reaction Planta Medica 61 466 - 469
Shim Y.H. , Choi J.H. , Park C.D. , Lim C.J. , Cho J.H. , Kim H.J. 2003 Molecular differentiation of Panax species by RAPD analysis Archives of Pharmacal Research 26 601 - 605
Taberlet P. , Gielly L. , Pautou G. , Bouvet J. 1991 Universal primers for amplification of three non-coding region of chloroplast DNA Plant Molecular Biology 17 1105 - 1109
Wang B. , Zhang C.G. , Gao P.F. , Wu X.M. , Zhao Y. 1996 Research progress on Nidus vespae, a traditional Chinese medicine derived from insents Journal of Pharmaceutical and Scientific Innovation 2 (6) 1 - 9
Wang J. , Ha W.Y. , Ngan F.N. , But P.P. , Shaw P.C. 2001 Application of sequence characterized amplified region (SCAR) analysis to authenticate Panax species and their adulterants Planta Medica 67 781 - 783
Zhang W.H. , Chen Z.D. , Li J.H. , Chen H.B. , Tang Y.C. 2003 Phylogeny of the Dipsacales s.I. based on chloroplast trnL-F and ndhF sequences Molecular Phylogenetics and Evolution 26 176 - 189
Zhu. S. , Fushimi H. , Cai S. , Komatsu K. 2004 Species identification from ginseng drugs by multiplex amplification refractory mutation system (MARMS) Planta Medica 70 189 - 192
Zhu. S. , Fushimi H. , Komatsu K. 2008 Development of a DNA microarray for authentication of ginseng drugs based on 18S rRNA gene sequence Journal of Agricultural and Food Chem. 3953-3959 56