Development of SCAR Markers Based on Improved RAPD Amplification Fragments and Molecular Cloning for Authentication of Herbal Medicines Angelica sinensis, Angelica acutiloba and Levisticum officinale

Molecular cloning from DNA fragments of improved RAPD amplification of Angelica sinensis, Angelica acutiloba and Levisticum officinale, provided novel sequence-characterized amplified region (SCAR) markers A13, A23, Al-34 and Al-0 whose sequences were deposited in the GenBank database with the accession numbers KP641315, KP641316, KP641317 and KP641318, respectively. By optional PCR amplification, the SCAR markers A13 and A23 are Levisticum officinale-specific, whereas the SCAR marker Al-34 is Angelica acutiloba-specific, and the SCAR marker Al-0 is Angelica sinensis-specific. These diagnostic SCAR markers may be useful for genetic authentications, for ecological conservation of all three medicinal plants and as a helpful tool for the genetic authentication of adulterant samples.

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Authentication of Herbal Medicines Dipsacus asper and Phlomoides umbrosa Using DNA Barcodes, Chloroplast Genome, and Sequence Characterized Amplified Region (SCAR) Marker

Molecules ◽

10.3390/molecules23071748 ◽

2018 ◽

Vol 23 (7) ◽

pp. 1748 ◽

Cited By ~ 8

Author(s):

Inkyu Park ◽

Sungyu Yang ◽

Wook Kim ◽

Pureum Noh ◽

Hyun Lee ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Scar Marker ◽

Herbal Medicines ◽

The Other ◽

Evolutionary Information ◽

Scar Markers ◽

Dna Barcodes ◽

Genomic Information ◽

Sequence Characterized Amplified Region ◽

Dipsacus Asper

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

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PCR-based molecular markers for identification of taxa from the Calypogeia fissa complex (Jungermanniopsida, Calypogeiaceae)

Biodiversity Research and Conservation ◽

10.2478/v10119-012-0024-3 ◽

2012 ◽

Vol 28 (1) ◽

pp. 9-18 ◽

Cited By ~ 1

Author(s):

Katarzyna Buczkowska ◽

Patrycja Gonera ◽

Bartosz Hornik

Keyword(s):

Molecular Markers ◽

Dna Sequences ◽

Scar Marker ◽

Issr Markers ◽

Genetic Differences ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Pcr Product ◽

Isozyme Loci ◽

Diagnostic Traits

Abstract Within Calypogeia fissa, two subspecies connected with geographic distribution are formally recognized: C. fissa subsp.fissa in Europe and C. fissa subsp.neogea in North America. Isoenzyme studies have shown that the European subspecies is genetically differentiated and composed of three genetically distinct groups PS, PB and G. The PS group has the most distinctive morphological features, but no morphological diagnostic traits have been found for groups PB and G. The sequence characterized amplified region (SCAR) markers developed on the basis of ISSR markers, applied in the study, allowed the delimitation of all groups distinguished in Europe within the C. fissa complex (PS, PB and G). The markers also revealed genetic differences between the European and American subspecies. Five primer pairs (Cal01, Cal03-Cal06) of the six pairs studied are useful as the diagnostic tool for the identification of particular groups from the C.fissa complex. The examined SCAR markers showed that the PS group of C.fissa subsp.fissa was the most distinct; it differed from both groups PB and G as well as from C.fissa subsp.neogea. All plants determined on the basis of diagnostic isozyme loci as the PS group amplified a longer product (380 bp) of the Cal04 primer pair than the rest of studied groups and yielded no amplification products in Cal03, Cal05 and Cal06 primers. The primer pair Cal03 distinguished the plants of the PB group from the remaining groups, since only the PB group generated a PCR product of about 290 bp. The genetic differences between all four studied groups of the C.fissa complex were supported by DNA sequences of the SCAR marker Cal04.

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Efficiency of Randomly Amplified Polymorphic DNA to Sequence Characterized Amplified Region Marker Conversion and their Comparative Polymerase Chain Reaction Sensitivity in Cucumber

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.124.2.128 ◽

1999 ◽

Vol 124 (2) ◽

pp. 128-135 ◽

Cited By ~ 30

Author(s):

Thomas Horejsi ◽

Jodie M. Box ◽

Jack E. Staub

Keyword(s):

Rapd Markers ◽

Scar Marker ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Pcr Product ◽

Rapd Pcr ◽

Pcr Products ◽

Polymerase Chain ◽

Template Dna ◽

Pcr Conditions

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

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Identification of the B, Q, and native Brazilian biotypes of the Bemisia tabaci species complex using Scar markers

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2016000500016 ◽

2016 ◽

Vol 51 (5) ◽

pp. 555-562 ◽

Cited By ~ 3

Author(s):

Paulo Roberto Queiroz ◽

Erica Soares Martins ◽

Nazaré Klautau ◽

Luzia Lima ◽

Lilian Praça ◽

...

Keyword(s):

Bemisia Tabaci ◽

Species Complex ◽

Scar Marker ◽

Random Amplified Polymorphic Dna ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Field Samples ◽

Scar Primer ◽

Q Biotype ◽

B Biotype

Abstract: The objective of this work was to develop sequence-characterized amplified region (Scar) markers to identify the B, Q, and native Brazilian biotypes of the sweet potato whitefly [Bemisia tabaci (Hemiptera: Aleyrodidae)]. Random amplified polymorphic DNA (RAPD) amplification products, exclusive to the B and Brazilian biotypes, were selected after the analysis of 12,000 samples, in order to design a specific Scar primer set. The BT-B1 and BT-B3 Scar markers, used to detect the B biotype, produced PCR fragments of 850 and 582 bp, respectively. The BT-BR1 Scar marker, used to identify the Brazilian biotype, produced a PCR fragment of 700 bp. The Scar markers were tested against the Q biotype, and a flowchart was proposed to indicate the decision steps to use these primers, in order to correctly discriminate the biotypes. This procedure allowed to identify the biotypes that occur in field samples, such as the B biotype. The used set of primers allowed to discriminate the B, Q, and native Brazilian biotypes of B. tabaci. These primers can be successfully used to identify the B biotype of B. tabaci from field samples, showing only one specific biotype present in all cultures.

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Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽

10.3390/molecules23092134 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2134 ◽

Cited By ~ 7

Author(s):

Pureum Noh ◽

Wook Kim ◽

Sungyu Yang ◽

Inkyu Park ◽

Byeong Moon

Keyword(s):

Herbal Medicines ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Its Region ◽

Sequencing Analysis ◽

Accurate Identification ◽

Republic Of China ◽

Sequence Characterized Amplified Region ◽

Angelicae Dahuricae Radix ◽

Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

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Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Revista de Biología Tropical ◽

10.15517/rbt.v68i1.37046 ◽

2020 ◽

Vol 68 (1) ◽

Cited By ~ 1

Author(s):

Zhiqiang Mei ◽

Md Asaduzzaman Khan ◽

Junjiang Fu

Keyword(s):

Random Amplified Polymorphic Dna ◽

Pcr Amplification ◽

Enzymatic Digestion ◽

Genetic Identification ◽

Perennial Herb ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Penthorum Chinense Pursh ◽

Living Organisms ◽

Penthorum Chinense

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random ampliﬁed polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh (P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

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Development of RAPD-SCAR markers for Lonicera japonica Thunb. (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning

Revista de Biología Tropical ◽

10.15517/rbt.v62i4.13493 ◽

2014 ◽

Vol 62 (4) ◽

pp. 1649 ◽

Cited By ~ 13

Author(s):

Luquan Yang ◽

Md. Asaduzzaman Khan ◽

Zhiqiang Mei ◽

Manman Yang ◽

Tiandan Zhang ◽

...

Keyword(s):

Molecular Markers ◽

Molecular Marker ◽

Scar Marker ◽

Rapd Analysis ◽

Random Amplified Polymorphic Dna ◽

Vital Role ◽

Lonicera Japonica ◽

Scar Markers ◽

Sequence Characterized Amplified Region ◽

Young Leaves

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved random ampliﬁed polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with different species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.de cualquier organismo, que hemos aplicado con éxito en L. japonica.

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Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo

Frontiers in Plant Science ◽

10.3389/fpls.2021.669458 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kaixin Zheng ◽

Yuchen Cai ◽

Weijie Chen ◽

Yadi Gao ◽

Jingjing Jin ◽

...

Keyword(s):

Scar Marker ◽

Pcr Amplification ◽

Distinct Species ◽

Start Codon ◽

Dendrobium Officinale ◽

Market Demand ◽

Medicinal Species ◽

Sequence Characterized Amplified Region ◽

Plant Resource ◽

Species Specific

The stems of Dendrobium officinale have been used as a rare and valuable Chinese tonic medicine, known as “Tiepi Fengdou”, since the Qing dynasty. Because of the increased market demand and continued exploitation of this plant, the reserves of wild D. officinale resources have been depleted, and D. officinale products on the market are being increasingly adulterated. Such changes have strongly affected the sustainable utilization of this valuable medicinal plant resource and the development of related industries. In this study, a species-specific DNA marker was developed for the rapid and accurate authentication of D. officinale. In total, 36 start codon-targeted (SCoT) polymorphism primers were screened in 36 definite Dendrobium species, and a distinct species-specific DNA amplicon (SCoT13-215) for D. officinale was obtained. After the sequence was cloned and sequenced, a sequence-characterized amplified region marker was developed (named SHF/SHR) and validated through PCR amplification of all 38 Dendrobium samples. The marker’s specificity for D. officinale was confirmed through the consistent amplification of a clear 197-bp band. This SCAR marker can be used to rapidly, effectively, and reliably identify D. officinale among various Dendrobium species and may play an important role in ensuring the quality of medicinal preparations and protecting the germplasm of this important medicinal species.

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