Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 Biomolecular Approach To Oligochaete Taxonomy Dr. Jaya. M 1* ,Dr. Aja. M2 and Dr. K. Vijayakumaran Nair3 1* Assistant Professor, Department of Zoology, Sree keralavarma College, Thrissur, Email: [email protected] 2Senior Research Fellow, Department of Zoology, University of Kerala, Kariavatom, Email: [email protected] 3Associate Professor, Department of Zoology, Mar Ivanios College, Thiruvananthapuram, Email: [email protected] ABSTRACT This paper comprises the molecular approach for the identification of earthworm along with the traditional taxonomic method. The mitochondrial CO 1 gene of the Pontoscolex corethrurus (Glossoscolecidae), Travoscolides chengannures, Amynthas corticis, Perionyx sansibaricus (Megascolecidae), Progizzardus varadiamensis and Glyphidrilus annandalei (Almidae) were sequenced. The cytochrome-c oxidase I (CO1) exhibited a unique barcode to a particular species. The further exploration of mitochondrial diversity in earthworms will lead to major improvements in our understanding of the evolutionary pathways and rates of the mitochondrial genome Key words: Barcoding, cytochrome c oxidase 1 (COI), 16S ribosomal DNA INTRODUCTION DNA barcoding is a taxonomic method that uses a short genetic marker in the DNA to identify an organism. It differs from molecular phylogeny in that the main goal is to identify an unknown sample in terms of a known classification [26]. DNA sequence can be used to identify different species, in the same way as the supermarket scanner uses the black stripes of the UPC barcode to identify the items. This database will rapidly link a specimen to a Binomial Linnaean name and through that link it will provide all available information and studies on the species. Moreover by allowing easy access to species identity, these tags would greatly enhance the rate of species discovery and further supplement and strengthen the traditional taxonomy. Most eukaryote cells contain mitochondria, and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variation in mtDNA sequences. This evolves much more rapidly than nuclear DNA, thereby resulting in the accumulation of differences between closely related species [6], [30] and [31]. This is more pronounced interspecifically (between species) than intraspecifically (within species). The DNA barcode itself consists of a 648 bp region, 58-705 from the 5’-end of the cytochrome c oxidase 1 (COI) gene using the mouse mitochondrial genome as a reference. It is based on the postulate that every species will most likely have a unique DNA barcode. There are 4650 possible ATGC-combinations compared to an estimated 10 million species remaining to be discovered [53]. It is also on the assumption that interspecific genetic variation exceeds intraspecific genetic variation [19], [21]. [37] reported that COI barcodes are also a good proxy for estimating intrageneric (within genus) phylogenetic diversity and relationships in earthworms. Early history of DNA barcode DNA barcode approach has become increasingly popular and the idea of a standardized molecular identification system emerged progressively during the 1990s with the development of PCR-based approaches for species identification. Molecular identification has largely been applied to bacterial studies, microbial diversity surveys [56] and [57] and routine pathogenic strains diagnoses [47] and [55] due to a need for culture-independent identification systems. PCR-based methods have also been frequently used in fields related to taxonomy, food and forensic molecular identification [50] and for identification of eukaryotic pathogens and vectors [28]. The Barcode of Life project aimed to create a universal system for a eukaryotic species inventory based on a standard molecular approach. It Available online @ www.ijntse.com 74 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 was initiated in 2003 by researchers at the University of Guelph in Ontario, Canada (http://www.barcoding.si.edu) and promoted in 2004 by the international initiative ‘Consortium for the Barcode of Life’ (CBOL). By then, it had more than 150 member organizations from 45 countries including natural history museums, zoos, herbaria, botanical gardens, university departments as well as private companies and governmental organizations. The DNA barcode project does not have the ambition to build the tree of life nor to perform molecular taxonomy [12] and [17], but rather to produce a simple diagnostic tool based on strong taxonomic knowledge that is collated in the DNA barcode reference library [44]. The DNA Barcode of Life Data System (BOLD, http://www.boldsystems.org) has progressively been developed since 2004 and was officially established in 2007 [41]. This data system enables the acquisition, storage, analysis and publication of DNA barcode records. During the year 2008, the total available DNA Barcode records were 363,584 sequences for 50,039 species. The efficiency of DNA barcoding has also been reported in the detection and description of new cryptic species [2], [7], [16], [24], [38], [49] and of sibling species [1]. DNA barcodes in biological specimens Until now, biological specimens were identified using morphological and anatomical features. If a specimen is damaged or is in an immature stage of development, even experienced taxonomist may be unable to make identifications. Barcoding solves these problems, because non-specialists can obtain barcodes from tiny amounts of tissue. This is not to say that traditional taxonomy has become less important, but rather that DNA barcoding can serve a dual purpose as a new tool in the taxonomists toolbox supplementing his knowledge as well as being an innovative device for researchers who need to make a quick (CBOL) identification [8]. DNA barcode is a powerful tool for identifying species of earthworms and provides a useful complement to traditional morphological taxonomy [23]. DNA barcode used in earthworm identification DNA barcoding promises great advances in species discovery and broad dissemination of taxonomic knowledge. This provides a framework for a first-stage taxonomic screening of earthworm diversity using a single standardised gene marker- a 648 base pair fragment of the mitochondrial cytochrome c oxidase subunit 1 gene. Earthworm barcode of life is a recently launched campaign under the broad umbrella of barcode of life initiative. The earthworm barcoding campaign objective is to build an extensive library of genetic tags (CO 1 barcode region- 648bp) for 5000 species. This also helps to discriminate morphologically similar and genetically different species termed cryptic species [9], [10], [11] and [35] by using the DNA sequences of the mitochondrial cytochrome c oxidase subunit1 (COI). However, reports are available that several genes have been chosen for identifying the earthworm species through DNA barcoding such as mitochondrial cytochrome-c oxidase I, 18S, 28S and 16S ribosomal DNA [5], [27], [33], [36], [39], [40], and [42]. Among these, DNA barcodes based on a fragment of the COI and 16S rDNA genes have been demonstrated to work well for species identification for earthworms. The aim of the study is to determine whether the mtCOI barcode is able to discriminate species under consideration and also check whether these are in agreement with those obtained through traditional approaches. Materials and Methods Collection of specimens The study area comprises the five districts of Kerala state (Thiruvananthapuram, Idukki, Thrissur, Palakad and Wyanad). The study was carried out during the period of September 2010 to October 2011. The earthworms were collected from the field by digging and hand sorting and either brought to the laboratory for preservation or washed and preserved in absolute alcohol (100%) on the spot. A total of 27 earthworm samples were collected from different habitats such as grassy land, river bank, forest, banana plantation, vegetation sites, residential area, coconut plantation, hill region, and cultivated land. All species were serially numbered and assigned a unique code. The adult worms were Available online @ www.ijntse.com 75 Dr. Jaya M et. al. / International Journal of New Technologies in Science and Engineering Vol. 2, Issue 6,Dec 2015, ISSN 2349-0780 identified by using traditional taxonomic keys. The preserved samples (in absolute alcohol, 100%, three-in-one AR grade) were stored at 4oC until processing for DNA extraction. The samples (posterior-most portion of the preserved specimen) were then processed for DNA sequencing. A total of 27 samples were used for DNA barcoding. The voucher specimens are deposited in the Zoological Museum of the Department of Zoology, Mar Ivanios College, Thiruvananthapuram, Kerala. Extraction of DNA DNA sequencing was done at ‘Rajiv Gandhi Centre for Biotechnology’, Department of Biotechnology, Government of India, Thiruvanathapuram. About 25 mg tissue of the each worm was used to extract the DNA. DNA was extracted from the sample using Qiagen® Dneasy Kit. The extraction protocol involves the following steps: 1. 25 mg of tissue cut into small pieces was placed in a 1.5 ml microcentrifuge tube. 2. 180 μl of AL buffer and 20 μl proteinase K were added. It was then mixed by vortexing and was incubated at 55◦C in a water bath until the tissue was completely lysed. 3. 4 μl of RNAase A was added to this and mixed by vortexing. It was incubated at room temperature for 2 minutes. 4. After vortexing for 15 seconds 200 μl of AL buffer was added to the sample. It was again mixed thoroughly by vortexing and was incubated for 70◦C for 10 minutes. 5. 200 μl of ethanol (96 - 100%) was added to the sample and mixed thoroughly by vortexing. 6. The mixture was pipetted to the DNeasy spin column and placed in a 2ml tube. 7. It was centrifuged at 8000 rpm for one minute. The flow through and collection tube was discarded. 8. The DNeasy spin column was placed in a new 2 ml collection tube and 500 μl AW1 buffer was added. 9.