“Should go parallel way – DNA barcoding needs to go hand in glove with other suitable molecular biology and analytical chemistry tools.”
merely relying only on the genome-based authen- tication will be insufficient for quality control of herbal products. Characterization for morphological and biochemical traits will also continue to play its parallel role in identification and assessment of medicinal plants in herbal industry (DeSalle, 2006).
parts/tissues collected in a season-specific manner have been prescribed to be used for therapeutic purpose (Shukla et al., 2013). If the prescribed plant part is replaced by a non-prescribed part or by a prescribed part collected in the wrong season, then the therapeutic activity of the product might be compromised and DNA barcoding will fail to identify the adulteration respon- sible for lowering the quality of the product.
systems biology components encompassing genomics (DNA barcoding) and metabolomics (for active secondary metabolites) in a major way and supplemented with need-based use of transcriptomics [specific expression subset analysis (SESA)] and proteomics (specific proteome) tools.
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DNA barcoding aims to find a single sequence to identify all species. Barcoding is generating a global, open access library of reference barcode sequences, which enables nontax- onomists to identify specimens
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CTAB CTAB method (Doyle and Doyle 1987) was used for isolation of total genomic DNA from collected medicinal plants material
dry market samples DNA was extracted using different DNA precipitation methods viz., CTAB modified method (Barnwell et al, 1998), the method used by Warude et al. 2003) and using DNeasy Plant Minikit, Quiagen
herbal samples of these plants available in market were so degraded that retrieving of sequences of the barcode loci was difficult. Nevertheless, botanical identity of at least one accession of the 14 species could be deciphered by matching with the developed barcode accompanied/ followed by/with BLAST search.
DNA barcoding can be used for the assignment of unknown specimens to a taxonomic group, authentic identification of phytomedicinals, and in plant biodiversity conservation.
both chloroplast/nuclear regions are used as universal barcodes for the authentication of phytomedicinals
recent advance in genomics has further enhanced the progress in DNA barcoding of plants by the introduction of high-throughput techniques like next generation sequencing, which has paved the way for complete plastome sequencing that is now termed as super-barcodes.
phytomedicinals and herbal product integrity and authenticity through DNA barcoding with the goal of protecting consumers from potential health risks associated with product substitution and contamination.
evaluate the herbal product authenticity.
barcoding gap” between inter- versus intraspecific divergences.
DNA barcoding: a genomics-based modern tool for plant authentication
new tool called ‘DNA barcoding’, proposed by Hebert et al. (2003a), is a valuable addition to the taxonomic tool box.
short DNA sequences from the specified region of genome termed as DNA barcode for biological identification
This difference between inter- and intraspecific distances is known as the ‘DNA barcoding ga
the case of plants, successful PCR of barcoding regions is often inhibited by the presence of secondary metabolites. However, modifications in extraction methods, primer sequences and the use of an engineered polymerase can usually overcome such problems. The combining of barcodes from multiple loci has also been used successfully
Single-locus approach
matK is one of the most rapidly evolving coding regions of the plastid genome and is hypothetically the closest plant analogue to the mitochondrial gene cytochrome oxidase 1 (COI) used as the animal barcode (Hollingsworth et al., 2011).
especially in nonangiosperms and rapid rate of substitution, along with the rare presence of frameshift indels and a few cases of premature stop codons, prompted some researchers to suggest that matK may not be functional in some taxa (CBOL, 2009; Hidalgo et al., 2004; Kugita et al., 2003).
highly conserved chloroplast gene rbcL, which encodes the large subunit of ribulose-1,5-bisphos- phate carboxylase/oxygenase (RUBISCO),
approxi- mately 1430 base pairs in length, is free from length mutations except at the far 30 end and has a fairly conservative rate of evolution.
Iphylogenetic relationships below the family level is often poor (Doebley et al., 1990)..
This intergenic region consists of two evolutionarily distinct parts, that is the psbA 30UTR, which is vital for post-transcriptional regulation of psbA gene expression, and the psbA-trnH intergenic spacer (IGS), which is highly variable and easily employed along a wide range of land plants (CBOL, 2009; Kress et al., 2005; Liu et al., 2012a).complex architecture of trnH-psbA makes it difficult to use as an individual barcode
regions of the nuclear ribosomal cistron (18S-5.8S- 26S), the internal transcribed spacer (ITS) region is the most commonly sequenced region.
considered suitable for amplification and sequencing owing to its shorter length of the target region referred to as a mini-barcode . two spacers of this region ITS1 and ITS2. problem of paralogy due to the occurrence of divergent copies within the individuals, which can lead to misidentification of samples
the 5S rDNA IGS (a variable region), plastid protein coding (rpoB, rpoC1), plastid IGSs (atpF-H, psbK-I) and low copy number genes,
Multilocus/tiered approach
matK and rbcL
rbcL and trnH–psbA
nrITS/nrITS2)
expenses of using a three-loci combination for large data sets,
tiered approach, in case of medicinal plants, is based on the use of a common, easily amplifiable and aligned region such as rbcL that can act as a scaffold for placing data from a highly variable region such as ITS2. ITS2 is a preferred second-tier candidate for medicinal plants due to its high species resolution, its presence in the nuclear genome (that evolves at a different rate than the plastid genome) and its shorter sequence that enables higher recovery from processed plant materials found within herbal products (Newmaster et al., 2013)
Next-generation sequencing (NGS)
parallel with many algorithmic advances through de novo sequencing, targeted resequencing, RNA interface to metagenomics, the technique is now encompassing the Sanger sequencing platform in DNA barcoding (especially metabarcoding)
Amplicon sequencing using ion torrent or 454 technolo- gies could potentially recover all of the filler plant species.
Whole- chloroplast genome sequence of Ceratophyllum demersum obtained using Roche’s 454 platforms by Moore et al. (2007) provided strong support to data obtained through traditional taxonomy.
Real-time DNA barcode-based high-resolution melting curve analysis (Bar-HRM)
used for detection of contamination in herbal mixtures.
discrimi- nating DNA sequence variants based on the characteristics of thermal denaturation of the amplicons without sequencing or hybridization procedures (Wittwer, 2009).
fluorescence monitoring of the melting curve of the dsDNA caused by the release of intercalating dye SYBR Green I in a real-time PCR system. HRM analysis requires no manual post- PCR processing, is performed in a closed-tube system and has a low reaction cost relative to other methods used to study genetic variation.
pathogenic identification, food authenticity and biological diagnostics.
chloroplast region trnH-psbA to identify adulterants in traded saffron by obtaining melting curves for saffron and its adulterants
species identification has been successfully described by Kalivas et al. (2014),
wide range of plants of medicinal importance especially within closely related species employed in the herbal sector.
HRM conjugated with specific barcode regions such as mitochondrial DNA, 16S rDNA regions and microsatellite markers has also been shown to be capable of accurately identifying products in the food industry (Bosmali et al., 2012; Ganopoulos et al., 2011, 2013).
l medicine is procured from the markets in the form of dried or powdered plant parts. DNA barcoding has been found successful in identification of plants from the finished herbal product
authentic standards of plant material for fulfilling the goal of Herb-Bol (barcode of life) research programme in the coming years.
biological reference material (BRM) would provide users with a universal platform for reference
BRM herbal barcode library for testing bulk materials could provide a method for good manufacturing practices (GMP) of herbal.
http://www.nibsc.org/products/brm_product_catalogue.aspx
Problems
problem concerning the barcoding of herbal products is the use of only plastid barcode regions due to insufficient nucleotide sequence variability to distinguish among closely related species.
varied gene copy number and PCR bias (Fazekas et al., 2009)
certain limiting factors such as low PCR efficiency, gene deletion and inadequate variation, no single-locus barcode exists as a universal DNA barcode for plants.
plants are influenced by both, their genome and their environment. Plant metabolism (mainly secondary metabolism, which is mainly responsible for the medicinal properties) is dependent on its environment (Briskin, 2000).
References-
Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V. DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant biotechnology journal. 2016 Jan 1;14(1):8-21.
Lou SK, Wong KL, Li M, But PP, Tsui SK, Shaw PC. An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database). BMC genomics. 2010 Dec;11(1):402.available at : https://rdccm.cuhk.edu.hk/mherbsdb/
https://rdccm.cuhk.edu.hk/mherbsdb/
Sundari KR. DNA barcoding: a genomic-based tool for authentication of phytomedicinals and its products. Botanics: Targets and Therapy. 2015;2015(5):77-84.