Short Introduction of Dna Barcoding

International Journal of Research Available at https://edupediapublications.org/journals e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 Short Introduction of Dna Barcoding L. Sarvananda [email protected] ABSTRACT unknown sample in terms of a preexisting DNA barcoding is a system for fast and accurate species identification. It’s creates ecological system more accessible by using short DNA sequence instead of whole genome and is used for eukaryotes and prokaryotes. The short DNA sequence is generated from standard region of genome known as marker. This marker is different for various species like CO1 cytochrome c oxidase 1 for animals, matK and rbcL for plants and Internal Transcribed Spacer (ITS) for fungus. It has many uses in various fields such as agriculture, sustaining natural resources, protecting endangered species, water quality, preserving natural classification. Barcodes are used in an effort to identify whether unknown species in sample should be combined or separated. The most commonly used barcode region in animal is a segment of mitochondrial gene cytochrome oxidase I (COI) that approximately contains 600 base pairs. Applications include identifying plant leaves due to absences of flowers or fruit, helps to identifying larvae stages of insects, which may not have significant characters than adults and are less well known , identifying the nutrition level of an animals , and identifying products of herbal supplements , wood ,or skin and other animal parts[1]. resources, identification of medicinal plants. Key words: matK, ITS, rbcL, taxonomy mitochondrial gene, PCR primers INTRODUCTION specific locus should be standardized in DNA barcoding , present in most of the texa of interest and sequenceable without specific PCR primers , short time to be easily DNA barcoding is a taxonomic method in sequenced with current technology , and which a short genetic marker to identify DNA provide a large variation between species yet a to which organisms or a particular species it relatively less variation within a species. A belongs. It helps to identify an several loci set as standardized regions were selected by the respective committees. For Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 673 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals animal and many other eukaryotes, the plasticity for the characters employed for species recognition can concatenation of the rbcL and matK are leads to incorrect identifications. Second, the chloroplast morphologically mitochondrial COI genes gene [2]. plant, These genes are and genetic variability cryptic taxa, in which the are providing poor resolution for land plants, and common in many groups [5]. Third, since the could morphological keys are often effective only for fungi, the a particular life stage or gender, many internal transcribed spacer region. It can be individuals‟ species cannot be identified. applied in algae, animal and also fungi, perhaps Finally, to a lesser degree due to a lower incidence of represent a major advance; the use of keys often hybridization compared to higher plants [3]. demands in high level of expertise that Population genetic studies can be done by these misdiagnoses are common [6]. regions to be assessed that complement rbcL and matK. In techniques and have large numbers the modern interactive versions of specimens at their disposal when the DNA quality is a lesser concern, and high-quality DNA samples gives more accurate in barcoding techniques would depend. To improve species concepts, It‟s to be develop a more sophisticated approach to barcoding, which would ideally include sequences from multiple independent markers, a multi-locus barcode, and specific inference tools that could be used to be limits and identify genetic „gaps‟, and also improve the information base depend on cruder plastid and mitochondrial DNA Figure1: barcodes [4]. system DNA based Identification Molecular analyze data is says that several barcodes DNA regions are suitable in Maturase K gene (matK) plants. It has four significant limitations in The chloroplast maturase K gene (matK) is, identification of species. First, both phenotypic with the exception of some ferns, situated Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 674 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals within an intron of the trnK gene. The gene is Paeonia (Paeoniaceae)[7]. Only 600- approximately 1535 basepair long in monocots 800basepair regions of the matK gene are and is the only chloroplast-encoded group II utilized for DNA-barcoding purposes. The intron maturase. Universal primers situated in matK gene evolves fast (three times faster than the trnK gene are used to amplify the entire rbcL and atpB) and some studies suggest it can gene region for phylogenetic studies in orders effectively discriminate between species in the or families, but are sometimes effectively used angiosperm [8]. on genus or species level, i.e. in the genus Figure 2: The matK chloroplast coding region based on the schematic drawing of Wakasugiet al. (1998), Matsumoto et al. (1998), Shaw et al. (2005) and Barthet&Hilu (2007) relates to the ITS which in bacteria and archaea, Internal Transcribed Spacer (ITS) Refers to the insert DNA to be found between the small-subunit ribosomal and RNA large-subunit (rRNA) genes in the chromosome or the corresponding transcribed region the polycistronic rRNA of precursor in transcript. In bacteria and archaea, ITS issituated between the 16S and 23S rRNA genes [9]. Then again, while ITS2 created as an insertion that disturbed the familial 23S rRNA gene. In Bacteria and Archaea, theITStake place in one to several copies, as do the neighboring 16S and 23S genes. When there are multiple copies, these do not occur in line to one another. Fairly, they occur in discrete locations in the circular chromosome[10]. there are two ITS's in eukaryotes; ITS1 is In eukaryotes, genes encrypting ribosomal located genes, RNA and inserts occur in cycle repeats that are while ITS2 is between 5.8S and 26S (in plants, thousands of copies long, each separated by or 28S in animals and fungi) rRNA genes. ITS1 regions between 18S and 5.8S rRNA Available online: https://edupediapublications.org/journals/index.php/IJR/ of non-transcribed P a g e | 675 DNA International Journal of Research Available at https://edupediapublications.org/journals termed intergenic spacer (IGS) or non- rRNAgene, e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 the ITS2, the 26S or 28S transcribed spacer (NTS). rRNA gene, and finally the 3' ETS[11]. Each eukaryotic ribosomal cluster contains the During rRNA maturation, ETS and ITS pieces 5' external transcribed sequence (5' ETS), are eliminated as non-functional by-products of the 18S this maturation, they are rapidly degraded.[12] rRNA gene, the ITS1, the 5.8S Figure: Organization of the eukaryotic nuclear ribosomal DNA tandem repeats. Sequence evaluation of the ITS region is widely used phylogeny because  in taxonomy and molecular of several It has a high degree of variation even between closely related species. This can be favorable explained by the relatively low evolutionary properties: pressure acting on such non-coding spacer sequences [13],[14].  It is routinely amplified thanks to its small size associated to the availability of highly   conserved flanking sequences ; RBCL It is stress-free to discoverlevel from small The chloroplast gene rbcL, which codes for the quantities of DNA due to the high copy larger unit of Ribulose-1, 5-bisphosphate number of the rRNA clusters ; carboxylase (RuBisCO). Which broadly used as It undergoes rapid concerted evolution via an unequal crossing-over and gene conversion. of phylogenetic in plant This endorses intra-genomic homogeneity taxonomy[15].Ribulose-1,5-bisphosphate of the repeat units, even if high-throughput carboxylase/oxygenase, commonly known by sequencing showed the occurrence of the appropriate locus for abbreviations RuBisCO, analysis RuBPCase, frequent variations within plant species. Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 676 International Journal of Research Available at https://edupediapublications.org/journals e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 or RuBPco, is an enzyme involved in the first divergence is typically detected between such major step of carbon fixation, a process by organisms, suggesting that the barcode is which atmospheric carbon dioxide is converted effective. In most if not all seed plants, by plants and other photosynthetic organisms however, the rate of evolution of cox1 is very to energy-rich molecules such slow[20]. chemical terms, as glucose. In it catalyzes the carboxylation ofribulose-1, 5- bisphosphate (also known as RuBP). It is probably the most abundant enzyme on Earth [16][17]. Cytochrome c oxidase I Cytochrome c oxidase I (COX1) also known as mitochondrially encoded cytochrome c oxidase I (MT-CO1) is a protein that in humans is encoded by the MT-CO1 gene.[18] In other eukaryotes, the gene is called COX1, CO1, or COICytochrome c oxidase I is the main subunit of the cytochrome c oxidase complex It is a gene that is often used as a DNA barcode to identify animal species[19]. MT-CO1 gene sequence is suitable for this role Figure: Location of the MT-CO1 gene in the human mitochondrial genome. MT-CO1 is one of the three cytochrome c oxidase subunit mitochondrial genes (orange boxes). REVIEW OF LITERATURE because its mutation rate is often fast enough to distinguish closely related species and also Hebert et al., (2003) implies the employment of because its sequence is conserved among sequences of DNA for the diagnosis of a conspecifics. Contrary to the primary objection species. By their research they emphasize the raised by skeptics that MT-CO1 sequence use of mitochondrial gene cytochrome c differences are too small to be detected between oxidase I in the global identification system, to closely related species, more than 2% sequence understand the diversity of life and also to study Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 677 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals molecular evolution. They created three COI barcoding. profiles, for seven phyla of animals, for eight phylogenetic relation that species invariably for largest order of insects and three for two clustered within genera and genera clustered hundred closely allied species of lepidopterans within families. Lowenstein et al., (2010) has to provide an overview of COI diversity. They used DNA barcoding in the identification of demonstrated that difference in COI sequences commercial fishes (tuna) classified under the were sufficient to assign organisms to their genus Thunnusand addressed the issue of public taxonomic categories providing resolutions that health by estimating the level of mercury in this cannot be obtained through morphological specific species and also banned the trade of analysis. Joly et al., (2014) considered the certain critically endangered species that were several reviews on DNA barcoding and in the menu in certain sushi restaurants of New presents the potential uses of DNA barcoding in York. Nwaniet al., (2011) made an application eco-informatics, community ecology, invasive of DNA barcoding in order to obtain detailed species, macroevolution, trait evolution, food facts on the distribution of fresh-water fish webs, trophic interactions and spatial ecology. species in Nigeria. They also established a They suggested that DNA barcoding would also reference library for the use of information in lead us to understand interactions between biodiversity evaluation and conservation giving species and the flow of energy in a food web. the remark that DNA barcoding could also help Hebert et al., (2004) tested the effectiveness of in barcoding using the COI gene in 260 bird Triantafyllidiset species of North America for the purpose of diversity of fish species in four Greek lakes identification and discrimination. The large using DNA barcoding with an objective to COI sequence variation concluded that the conserve and manage the threatened species. variation within closely related species were They examined 37 species and found deep higher than variation the within species. divergences among the specimens collected food Their research al., results and showed market (2011) some analysis. analyzed the from the four lakes. De Carvalhoet al., (2011) Ward et al., (2005) barcoded 207 species of Australian Marine fishes and were able to differentiate one from the other by cox I studied the diversity of COI sequences among 100 species of fish in the Sao Francisco River basin to evaluate the efficacies of barcoding in the differentiation of species. They found deep Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 678 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals intra-specific divergences within nine species Hogg et al., (2007) evaluated the sequence and discovered several new species and genera. diversity in the mitochondrial cytochrome-c Luiz et al., (2011) proved the efficiency of oxidase 1 gene as a tool for resolving DNA barcoding in the detection of hidden differences among species of Arctic springtails. biodiversity in the Upper Parana Basin of Foottiet al., (2008) using DNA barcoding were Brazil. Their study showed that the fish able to discriminate nearly 300 species of Piabinaargentearepresented a minimum of five aphids (Hemiptera: Aphidae) and concluded new species of fish suggesting that geographic that 96% were well differentiated though isolation possibly enabled the formation of new sequence variation was low. species. The complex life cycles of the aphids and their parthenogenic mode of reproduction did not Ward et al., (2008) studied 191 species of prove to be a barrier. Sheffield et al., (2009) echinoderms that included five classes and used DNA barcoding which is a reliable and concluded the barcode sequence; COI that rapid means of species-level identification for comprises of 657bp is an effective, accurate and ecological studies of bee communities. Their useful method for the diagnosis of all the five work classes of Echinodermata. undescribed genetically unique species of bee. led to the identification of two Utsugiet al., (2011) with respect to the extreme Raduloviciet al., (2009) used DNA barcoding diversity of insects and their ecological, for metazoans epidemiological and agricultural importance including crustaceans in the Estuary and Gulf of imply that this technique of DNA barcoding has St Lawrence .They concluded that genetic attracted the attention of many taxonomists, distances between species were 25 times higher agriculturists, than within species. ecologists. Ander et al., (2012) sequenced and species identification of Their research also led to the identification of invasive Amphipods in St. Lawrence estuary marking the importance of this system of COI gene identification. conservation biologists and studied the COI gene of 237 specimens of biting midges (Culicoides), who were insect vectors of veterinary diseases and by their study found that Available online: https://edupediapublications.org/journals/index.php/IJR/ there was deep intraspecific P a g e | 679 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals divergence among the 237 specimens and were butterflies able to differentiate 95% of the species. Lycaenidaeand Ng'endoet al., mitochondrial (2013) DNA sequenced Cytochrome the oxidase subunit 1, COI gene from 47 ants of the genus Pheidole. Their work resulted in significant findings where most sequences clustered into well differentiated groups and the sequences in a cluster were quite distinct. Smith et al., (2013) used DNA barcoding and studied the identification of microgastrine wasps that are parasitoids of caterpillars, the host-parasitoid biology and also ecology. Because of their use as biological control agents the study resulted in biological control programmes, description of belonging found to the family that inter specific variation exceeds intraspecific variation and found the absence of a barcode gap. They were able to identify several cryptic species that which did not differ phenotypically from the others. They gave the impression that minimum distances between species are critical and not average distances. Their success rate was 58%.Hajibabaei et al., (2006) were able to effectively discriminate three families of tropical lepidopterans using the mitochondrial cytochrome c. Their result showed barcoding of the COI gene helped to distinguish 97.9% of the specimens. new cryptic species and taxa. Smith et al., Kronestedtet al., (2010) proposed a new genus (2014) performed barcoding on a group of Draposafor the former Genus Pardosathat arthropod, the ants of Madagascar, which consisted of eight species of wolf spiders in the exhibited hyper diversity. Indo-Malayan region. These high divergences furthermore paved the way for detailed genetic, morphological and behavioral studies. They barcoded 280 specimens belonging from 28 genera and derived vast diversity patterns across the locality of Madagascar. Kumar et al., (2007) barcoded DNA sequences of mitochondrial cytochrome oxidase gene and were able to identify 62 species out of 63 specimens. Wiemerset al., (2007) did a study on blue Barrett et al., (2005) worked on DNA based identification and established the potential of COI as a rapid and accurate identification tool for biodiversity survey of spiders. Astrinet al., (2006) worked on the identification of species using species identification methods COI and 16 sRNA. Binfordet al., (2008) studied that Phylogenetic relationships of Loxoscelesand Sicariusspiders are consistent with western GondwananVicarience Available online: https://edupediapublications.org/journals/index.php/IJR/ using the help P a g e | 680 of e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals molecular dating analyses of 28S, COI, 16S and Digeneanparasites of amphibians with an NADHI sequences. objective discovering new species and accurate They presented data which made evident that Loxoscelesand Sicariusspiders were very old and have diversified since the separation of African and South American continents. Robinson et al., (2009) has applied the knowledge of DNA barcoding for the discrimination. They described nearly five new species of frog lung flukes out of 13 species and explained that apart from morphological diagnosis, DNA barcoding of species further enhanced the identification of intraspecific specimens. identification of 19 species- rich genera of Guet al., (2011) used DNA barcoding for the spiders in order to find out the existence of a identification of the crude drug gecko, Gekko barcode gap. From their study, they deduced gecko which is valued as a traditional Chinese that values of divergences were quite variable medicine. As a result, the population of this among genera. Their study also revealed crude drug gecko was declining and DNA maximum and barcoding was done to identify the adulterants suggested the collaboration of molecular and used instead of the crude drug gecko. Nagy et morphological identification system for global al., (2013) did the first large scale reptile identification of spiders. barcoding assessment of entire reptile fauna of intraspecific divergence Peterson et al., (2007) worked on the identification of Mexican Tarantulas by the fourth largest island in the world, the Biodiversity Hotspots of Madagascar. investigating the mitochondrial DNA sequence They used newly designed reptile specific from the cytochrome - c oxidase subunit 1 gene. primers and identified nearly 40 new species of Identification of these species were important snakes, for International Wildlife Law enforcement and Meganathanet conservation, researches were done accurately knowledge without the death of other species. identification of threatened crocodile species in Regagnonet al., (2010) tested the potential of cox 1 gene in the discrimination of Helminthes (Platy helminthes: Digenea) that were skinks, of chameleons al., DNA (2013) and geckos. utilized barcoding for the the India due to illegal hunting. They adopted DNA barcoding of the barcode region that consisted of 750bp in crocodiles in their conservation strategy. Venceset al., (2014) used DNA Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 681 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals barcoding to understand the genetic variation in monitoring water quality, authentication of two widespread skinks from Madagascar, natural health products and identification of Trachylepiselegansand medicinal plants. Trachylepisgravenhorstii. Although both the species were morphologically except well-differentiated for some slight differences DNA barcoding proved to be a promising tool for identification. Controlling Agricultural Pest: DNA barcoding can help in identifying pests in any stage of life making easier to control them saving farmers from cost of billion dollars from pest damage. The global Clare set al., (2007) sequenced the cytochrome c oxidase subunit gene 1 in 87 species of bats from Guyana for the sole purpose of studying tephritid barcoding initiative contributes to management of fruit flies by providing tools to identify and stop fruit flies at border. diversity. 81% of the species showed low Identifying intraspecific variation and their sequences barcoding allows non ecologists to identify showed clear divergences. Six species of bats the vector species that can cause serious showed deep intraspecific divergences. Muller infectious diseases to animals and humans, et al., (2013) utilized the technique of to understand these diseases and cure them. barcoding in relation to human health issues so A global mosquito barcoding initiative in that it could aid in the identification and correct building a reference barcode library that can elimination of rodents classified under the help public health officials to control these subfamily Sigmodontidae. This was done in diseases order to reduce the risk of transmission of the effectively and with very less use of hanta virus through these rodent species as they insecticides. are the reservoirs of these viruses. vector species DNA more barcoding, natural resource managers can DNA barcoding has many applications in various fields like preserving natural resources, endangered causing Vectors: Sustaining Natural Resources: Using DNA APPLICATIONS protecting Disease species, controlling monitor illegal trade of products made of natural resources like hardwood trees. Fishbol is reference barcode library for agriculture pests, identifying disease vectors, Available online: https://edupediapublications.org/journals/index.php/IJR/ P a g e | 682 e-ISSN: 2348-6848 p-ISSN: 2348-795X Volume 05 Issue 04 February 2018 International Journal of Research Available at https://edupediapublications.org/journals hardwood trees to improve management and conservation of natural resources. Protecting Endangered Species: Primate Population is reduced in Africa by 90% because of bush meat hunting. DNA barcoding can be used by law enforcement to bush meat in local markets which is obtained from bush meat. Identification of medical plants [21]. CONCLUSION DNA barcoding is a system for fast and accurate species identification which will make ecological system more accessible. It has many applications in various fields like controlling agricultural pests, sustaining natural resources, protecting endangered species, monitoring Monitoring Water Quality: Drinking water water quality, preserving natural resources, is a process resource for living being. By protecting studying organism living in lakes, rivers and identification of medicinal plants. endangered species and streams, their health can be measured or determined. DNA barcoding is used to create a library of these species that can be difficult to identify. Barcoding can be used by environmental agencies to improve determination of quality and to create better policies which can ensure safe supply of drinking water. REFERENCES [1] Paul D. N. Hebert, AlinaCywinska, Shelley L. Balland Jeremy R. deWaard. “Biological identifications through DNA barcodes”. Proceedings of the royal society, 2010; 313 – 321. Routine Authentication of Natural Health [2] Products: Authenticity of natural health Wilkinson, James M. Dunwell, Rao Prasad products is an important legal, economic, Kesanakurthi, Nadia Haidar and Vincent health and conservation issue. Natural Savolainen. “Land plants and DNA barcodes: health products are often considered as safe short-term and long-term goals”. Phil. Trans. because of their natural origin. R. Soc. B 360, 2005; 1889–1895. Identifying of plant leaves even if flowers or fruit are not available. [3] Mark W. 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