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PHYLOGENETIC ANALYSIS of FRESHWATER MUSSEL Corbicula Regularis by 18S Rrna GENE SEQUENCING

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PHYLOGENETIC ANALYSIS of FRESHWATER MUSSEL Corbicula Regularis by 18S Rrna GENE SEQUENCING Journal of Experimental Biology and Agricultural Sciences, April - 2015; Volume – 3(2) Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org ISSN No. 2320 – 8694 PHYLOGENETIC ANALYSIS OF FRESHWATER MUSSEL Corbicula regularis BY 18S rRNA GENE SEQUENCING Magare V N1, Kulkarnii C P2, Maurya C B3 , Patil R C4,* and Upadhye M V5 1Principal, Department of Zoology, Kirti M. Doongursee College of Arts, Science and Commerce, Dadar (W), Mumbai- 400028, INDIA 2Department of Chemistry, Kirti M. Doongursee College of Arts, Science and Commerce, Dadar (W), Mumbai - 400028, INDIA 3Department of Chemistry, G.N. Khalsa College of Arts, Science & Commerce, Matunga (E), Mumbai - 400019, INDIA 4Department of Microbiology, Bhavan’s College, Andheri (W), Mumbai - 400058, INDIA 5Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai - 400098, INDIA Received – April 05, 2015; Revision – April 15, 2015; Accepted – April 25, 2015 Available Online – April 25, 2015 DOI: http://dx.doi.org10.18006/2015.3(2).213.219 KEYWORDS ABSTRACT Phylogenetics Corbicula regularis is a freshwater mussel found in the Indian sub-continent. In the present study, 18S ribosomal gene phylogenetic characterization of this important bivalve was attempted using 18S ribosomal RNA gene markers. Genomic DNA was extracted and 18S rRNA gene was amplified by universal primers. The Corbicula regularis amplification product was sequenced and compared with the nucleotide databases available online to evaluate phylogenetic relationship of the animal under study. Results indicated that 18S rRNA gene Freshwater mussels sequences of C. regularis showed high degree of similarity to another freshwater mussel, C. fluminea. This work constitutes the first ever sequence deposition of the C. regularis in the nucleotide databases State of Maharashtra highlighting the usefulness of 18S ribosomal gene markers for phylogenetic analysis. All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. * Corresponding author E-mail: [email protected] (Patil R C) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher (www.my-vision.webs.com/horizon.html). All _________________________________________________________rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org 214 Patil et al 1 Introduction and different body parts were carefully dissected and separated. Freshly dissected body parts were subjected to Freshwater mussels are one of the most important components isolation of genomic DNA. of freshwater aquatic ecosystem. These shellfish are filter feeders that siphon phytoplankton and organic detritus 2.2 Isolation of Genomic DNA suspended or dissolved in the water column. In this manner, they serve as indicators of ecosystem health and help to sustain Genomic DNA from mantle tissue of the bivalve was extracted the water quality (Goudreau et al., 1993). Apart from this role, by following the protocol standardized by Upadhye et al. like other bivalves, they also serve as food for benthic (2011). Briefly, 100 mg fresh mantle tissue was ground in 2 ml invertebrates, fish, birds and some mammals (Langdon & of extraction buffer by adding buffer in parts to obtain a Newell, 1996). They have also been used for making buttons, homogenous suspension. This suspension was centrifuged at as food, source material for tools and ornamental objects and 10000 rpm for 5 min and supernatant was discarded. To the recently for cultured pearl production (Upadhye et al., 2011). pellet, 700 µl of suspension buffer and 80 µl of SDS were added and incubated at 65°C for 30 min. To this mixture 200 Though India has a rich diversity of freshwater pearly mussels µl ammonium acetate was added and incubated for another 30 with a widespread distribution of around 52 different species of min. After incubation, 500 µl chloroform - isoamyl alcohol freshwater mussels; the marine molluscs have received more (24:1) mixture was added and mixed it by several inversions. attention because of their aesthetic and gastronomic appeals This mixture was centrifuged at 10000 rpm for 8 min. Top and the use of freshwater mussels for commercial applications aqueous layer was collected in a new microfuge tube and equal is in infancy (Subba Rao, 1993). In India, in comparison to volume of absolute ethanol was added. This mixture was kept marine mussels, freshwater molluscs are drab coloured and in ice cold condition for 1 hour and after incubation centrifuge have attracted less attention. Notwithstanding their importance at 10000 rpm for 15 min. The pellet was dissolved in 200 µl in the ecosystem, freshwater molluscs are less explored and elution buffer and stored at 4°C. Agarose gel electrophoresis studied. (1.0 % agarose) was carried out to check the presence and quality of DNA. C. regularis is an important member of this Indian freshwater mussel fauna. This bivalve belongs to family Corbiculidae and 2.3 18S rRNA Gene Amplification using PCR was earlier reported to be quite common all over the country (Subba Rao, 1989). However, due to increasing anthropogenic Isolated bivalve DNA was used for phylogenetic analysis. PCR activities and pollution resulting in habitat destruction, this amplification was carried out with reaction mixture of 50 µl mussel has disappeared from many locations where it was containing was 25 µl of PCR master mix, 1 µl of template earlier used to be abundantly present. Work done on this DNA (diluted to 10-50 ng/µl), 1.2 µl of forward primer UnivF- freshwater bivalve has been restricted to studying its 15 (5’ CTG CCA GTA GTC ATA TGC) (10 µM), 1.2 µl of physiological, biochemical and toxicological aspects reverse primer Univ R-1765 (5’ ACC TTG TTA CGA CTT (Mudkhede & Nagabhushanam, 1977; Lomte & Jadhav, 1982; TAC) (10 µM) and 8.1 µl of nuclease free water in a PCR tube Mali & Afsar, 2010) and there are absolutely no reports in Biometra thermal cycler. PCR conditions were as follows: available on any scientific study on this mussel with the aid of initial denaturation at 95°C for 2 min, followed by 30 cycles of modern sophisticated molecular biological tools. each 95°C for 1 min, primer annealing at 52°C for 1 min and final primer extension at 72°C for 5 min. Amplified gene Considering the fact that its resources are getting depleted products were visualized on 1% agarose gel under UV rapidly and this important mussel could soon be on the verge visibility @ 254 nm. PCR products were purified with a kit of getting endangered, an attempt was made in the present and sequenced by using forward and reverse sequencing study to characterize C. regularis using amplified sequence of primers. DNA sequencing was performed on automatic DNA its 18S ribosomal RNA gene and study its phylogenetic sequencer supplied by Applied Biosystems using BigDye position with respect to available gene sequences of other Terminator v 3.1 Cycle Sequencing Kit. mussels. 2.4 BLAST and Phylogenetic analysis 2 Materials and Methods The amplified PCR product was partially sequenced and the 2.1 Sample collection gene sequences were analyzed against the DNA database of Japan (DDBJ) database using BLAST N (nucleotide- Thirty healthy specimens of C. regularis were hand collected nucleotide BLAST) program packages (Zhang et al., 2000) and from Darna river near Nashik city in the State of Maharashtra. matched to known 18S rRNA gene sequences. After Collected samples were brought alive in plastic containers to identifying the phylogenetic neighbors of the study animal, the the Department of Zoology, Kirti M. Doongursee College, sequences were also analyzed by using CLUSTAL W and Mumbai and acclimatized for 24 hours in separate glass Phylodraw softwares (Choi et al., 2000). Distance between the aquaria. After acclimatization, 15 live mussels were sacrificed genes for each pair was found out and phylogenetic tree was _________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org Phylogenetic analysis of freshwater mussel corbicula regularis by 18s rRNA gene sequencing., 215 constructed. Sequence of C. regularis was submitted to DNA morphological data matrices. Since last couple of decades, Database of Japan (DDBJ). phylogeny studies of various molluscan taxa with the aid of ribosomal marker gene have been a reliable tool in elucidating 2.5 Comparison of sequence with other freshwater mussels of the phyletic relationships below the class level (Taylor et al., Maharashtra 2007a; Taylor et al.,2007b). However, with only couple of report by Jadhav and Jamkhedkar (2009) who studied The 18S rRNA gene sequence of C. regularis was analyzed by phylogeny of freshwater mussel, L. corrianus, and that of Clustal W software to find out the pair distance between 18S Upadhye et al., (2011) who studied phylogeny of freshwater rRNA gene sequence of other freshwater mussels present in the mussel, P. corrugata there are absolutely no other reports Maharashtra state such as Parreysia corrugata (accession available on DNA sequencing of freshwater mussels from the number AB533279) and Lamellidens corrianus (accession state of Maharashtra so the present study was undertaken. number EU394233). Phylogenetic tree was constructed for these three species using Phylodraw software. The 18S rRNA gene of freshwater
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