Species-specific Sequence Characterized Amplified Region Markers Research Articles

Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines.

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

Mei Z, Khan MA, Zhang X, Fu J. 2017. Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers. Biodiversitas 18: 1243-1249. For the genetic identification and authentication of living organisms, development of Sequence-Characterized Amplified Region (SCAR) markers from Random Amplified Polymorphic DNA (RAPD) fragments is a valuable molecular approach. By using SCAR markers, molecular analysis is simplified to a Polymerase Chain Reaction (PCR) analysis using PCR primers designed from specific sequences of the RAPD amplicons. In this study, RAPD fragments from improved RAPD amplification of a perennial herb Penthorum chinense Pursh from China were cloned into a T-vector, and positive clones were identified by PCR amplification, and sequenced with the Sanger sequencing method for the SCAR marker development. Five SCAR markers were developed that were very specific to P. chinense, and deposited in GenBank (accession numbers: KX671029, KX671030, KX671031, KX671032 and KX671033). BLAST searches of these five nucleotide sequences in the GenBank database showed no identity with markers from other species. This study was developing five specific SCAR markers, has enabled reliable genetic identification of the herbal plant species Penthorum chinense Pursh, useful for authenticating future samples of this important medicinal herb.

Development of molecular markers for authentication of the medicinal plant species Patrinia by random amplified polymorphic DNA (RAPD) analysis and multiplex-PCR

The authentication of components from plant species is vital for the quality control and standardization of herbal medicines. Due to morphological similarities when dried, it is very difficult to distinguish authentic medicinal plants Patrinia villosa and P. scabiosifolia from adulterants of P. rupestris and P. saniculifolia in the herbal medicine Patriniae Radix. To establish a reliable authentication tool distinguishing authentic Patriniae Radix species from adulterants, we used random amplified polymorphic DNA (RAPD) genomic profiling and obtained several species-specific DNA fragments for each of the four Patrinia species. Based on the sequences of these amplicons, we developed sequence characterized amplified region (SCAR) markers as stable molecular authentication tools. SCAR markers to identify the authentic Patriniae Radix species, P. villosa and P. scabiosifolia, were developed. To accurately identify adulterants, we also developed SCAR markers able to identify the inauthentic P. rupestris and P. saniculifolia species. Furthermore, we established multiplex-PCR SCAR assays to simultaneously distinguish all four species using combinations of species-specific SCAR marker primer sets. These efforts resulted in reliable DNA-based molecular markers to identify authentic Patriniae Radix species from their adulterants and a rapid authentication assay for the efficient identification of Patrinia species. These markers will facilitate the standardization of Patriniae Radix preparations and therefore prevent the distribution of adulterants.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities.

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

Molecular identification of lactic acid bacteria in Chinese rice wine using species-specific multiplex PCR

Chinese rice wine (CRW), a national unique and traditional food, has more than 4,000 years of history and is very popular in China. Lactic acid bacteria (LAB) play important roles in the fermentation of CRW because of its strong lactic acid production and high acid tolerance. It would be of practical interest to isolate the LAB species from CRW and to develop a simple and effective method for its accurate identification. In this study, 11 CRW-related LAB strains, frequently occurring in the fermentation of CRW, were isolated or collected and analyzed with the nested specifically amplified polymorphic DNA (nSAPD)-PCR technique. Eleven species-specific sequence characterized amplified region (SCAR) markers were generated from 11 polymorphic nSAPD bands. Three multiplex PCR assays were further established for the joint use of the 11 species-specific SCAR markers. Thus, a simple rapid molecular method was developed based on the application of nSAPD, SCAR, and multiplex PCR techniques, which allowed a rapid and simultaneous detection of 11 CRW-related LAB species. The simple rapid PCR-based molecular method can be used as a useful tool for the identification of LAB species in the traditionally fermented foods, especially in the rapid developing CRW industry in China.

Molecular characterization of Eimeria spp. from chicken by Polymerase Chain Reaction based on species-specific SCAR markers

Coccidiosis in chickens is one of the major problems of poultry industry that is caused by protozoan parasites of genus Eimeria . Present study was conducted to characterize the Eimeria species infecting poultry in different regions of Chittagong district of Bangladesh. Seven species of Eimeria including E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, E. mitis, and E. praecox have been characterized using conventional PCR analysis through amplifying unique single copy sequences derived from sequence characterized amplified region (SCAR) markers. The present study is the first of its type to use molecular tools to identify Eimeria spp. infecting chicken in Bangladesh. The study indicates that the modern molecular technique involving PCR to detect the Eimeria species are more reliable and accurate than the traditional morphology-based technique.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

Development of a species-specific sequence-characterized amplified region marker for roses

DNA fingerprints of four rose species, Rosa centifolia, R. Gruss-an-Teplitz, R. bourboniana, and R. damascena, were developed using RAPD-PCR. We identified a unique polymorphic band in R. centifolia. This 762-bp fragment was produced by the random primer GLI-2. The fragment was eluted and directly cloned in a TA cloning vector, pTZ57R/T. Digestion of the plasmid with EcoRI confirmed the cloning of GLI-2(762) in pTZ57R/T. A second enzyme, PstI, used in combination with EcoRI, gave complete digestion of the plasmid, and the 762-bp fragment was confirmed on the gel. Subsequently, the polymorphic amplicon was sequenced with an AB1 373 DNA sequencer system using the PRISM(TM) Ready Reaction DyeDeoxy(TM) Terminator Cycle Sequencing kit. After sequencing, specific primers (23 bp long) were designed based on the sequence of the flanking regions of the original RAPD fragment. These primers will effectively allow fingerprinting for the identification of R. centifolia species. In essence, we developed an SCAR marker to authenticate the identity of R. centifolia species and to distinguish it from its substitutes. Such techniques are required not only to complement conventional parameters in creating the passport data of commercial and medicinal products of rose, but also for routine quality control in commercial and government rosaries and rose nurseries.

The development of species-specific SCAR markers for delimitation of Calypogeia species

The genus Calypogeia belongs to one of the most difficult groups of leafy liverworts, with about 90 species. Nine species of the genus are currently known from Europe: C. azurea, C. integristipula, C. neesiana, C. suecica, C. muelleriana, C. sphagnicola, C. fissa, C. arguta, and C. azorica. Recently, another species, morphologically resembling C. muelleriana but genetically distinct from it, was detected using isozyme markers. In the present study, C. muelleriana and C. sp. nov. were further characterized by 13 and 14 specific inter-simple sequence repeat band patterns, respectively. Nei’s genetic distance between the two species was 0·61. Three species-specific bands were converted into sequence characterized amplified region (SCAR) markers. The first SCAR primer pair amplified a product of 360 and 600 bp in C. muelleriana and C. sp. nov., respectively. The two other SCAR markers were diagnostic for other European Calypogeia species.

Molecular Fingerprinting and Population Dynamics of Brevipalpus Mites on Texas Citrus

Abstract Three species of Brevipalpus mites are known potential vectors of the citrus leprosis virus (family Rhabdoviridae, genus Cyto- and Nucleorhabdovirus, CiLV). Species identification of these mites is difficult because of their small size and morphological similarities. The objective of this study was to develop an accurate and rapid molecular fingerprinting method for identifying B. phoenicis and B. californicus on Texas citrus. Iso-colonies of the two Brevipalpus species were cultured on immature citrus fruit and identified using a dichotomous key. Whole genome amplification was used to produce DNA template from single mites to identify the two species by amplified fragment length polymorphism and sequence-characterized amplified region (SCAR). Population dynamics of Brevipalpus were monitored in four citrus orchards during the 2007 growing season. Subsamples of mites were identified morphologically or by species-specific SCAR markers. Molecular fingerprinting was very efficient in identifying the two species of mites in subsamples, even in cases where the morphological identification did not discriminate between species. Brevipalpus spp. density per fruit was significantly affected by the host plant species, with sweet orange fruit hosting significantly higher density than grapefruit, Citrus x paradisi Macfad. The percentage of fruit infested with Brevipalpus mites varied significantly with time but not with host plant and the host plant by sampling date interaction. Comparison of molecular fingerprinting and morphological identification provided a high percentage of match >85% between the two methods, suggesting that molecular fingerprinting could facilitate studies on population dynamics of Brevipalpus in citrus.

Development of SCAR Markers for Species Identification of the Genus Nepenthes (Nepenthaceae)

Nepenthes species in Thailand, namely N. mirabilis Druce, N. gracilis Korth., N. smilesii Hemsl., N. ampullaria Jack and N. kampotiana Lecomte, were collected for development of Sequence Characterized Amplified Region (SCAR) marker, a genotype identification tool. Forty Random Amplified Polymorphic DNA (RAPD) primers were screened and three successful primers produced different banding patterns including five candidate species-specific markers. The candidate markers were cloned and sequenced. The marker sequences are 602, 379, 420, 473 and 1017 bp for N. mirabilis, N. gracilis, N. smilesii, N. ampullaria and N. kampotiana, respectively. Then the sequences were used to design primers for development of a species-specific band being a SCAR marker, including Mir 1, Mir 2 and Mir 3 for N. mirabilis; Gra 1 and Gra 2 for N. gracilis; Smi 1, Smi 2 and Smi 3 for N. smilesii; Amp 1 and Amp 2 for N. ampullaria and Kam 1 and Kam 2 of N. kampotiana. The primers were evaluated with each other Nepenthes species. Finally, species-specific SCAR markers were successfully developed for N. gracilis, N. ampullaria and N. kampotiana. Application of these markers is feasible for identification of Nepenthes species in Thailand.

The application of DGGE and AFLP-derived SCAR for discrimination between Atlantic salmon ( Salmo salar) and rainbow trout ( Oncorhynchus mykiss)

In an attempt to authenticate commercial frauds in fresh products of Atlantic salmon ( Salmo salar) by being adulterated and substituted with those of rainbow trout ( Oncorhynchus mykiss), two molecular tools, AFLP (amplified fragment length polymorphism) and DGGE (denaturing gradient gel electrophoresis) were explored here. A species-specific SCAR (sequence characterized amplified region) marker for rainbow trout ( O. mykiss) was developed based on AFLP analysis. In addition, DGGE was utilized to discriminate Atlantic salmon ( S. salar) from rainbow trout employing a fragment of the mitochondrial cytochrome b gene. Analysis of experimental mixtures demonstrated that the detection sensitivity of AFLP-derived SCAR is higher than that of DGGE. In order to ascertain commercial frauds are fabricated by the adulteration or substitution of rainbow trout, DGGE analysis of cytochrome b gene should be combined because only one species-specific SCAR just for rainbow trout was obtained in this study.

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo.

The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.

Development of Species-Specific SCAR Markers in Bentgrass

Bentgrass species (Agrostis spp.) are cool season turfgrasses that are tolerant of continuous, close mowing heights because of their prostrate growth habit. Some bentgrass species are difficult to distinguish because of similar morphological characteristics and genetic compatibility. Specific DNA technology, such as the use of SCAR (sequence characterized amplified region) markers, can be used to differentiate between some species of bentgrass. SCAR markers were created by sequencing a single RAPD (random amplified polymorphic DNA) band and designing primers to amplify the band of specific size. Two SCAR primer pairs were designed to identify colonial (Agrostis capillaris L.) and creeping (Agrostis palustris Huds.) bentgrass species. The colonial SCAR primers amplify a band of 400 base pairs (bp) and are designated Col400F and Col400R. The creeping SCAR primers amplify a band of 700 bp and are designated Creep700F and Creep700R. Testing with 17 cultivars (140 plants) representing four bentgrass species, single unique bands were correctly amplified for creeping and colonial bentgrass species by their respective SCAR primer pair. Differentiating these species by SCAR markers is useful for screening large numbers of clones collected from superior patches of naturalized populations on old golf courses and lawns. These SCAR markers also have useful potential for identifying progenies derived from artificial interspecific hybridizations among bentgrass species, especially between colonial and creeping bentgrass.