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Phylogenetic Analysis of 18S Rdna of Freshwater Copepods Neodiaptomus Species and Mesocyclops Species

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Phylogenetic Analysis of 18S Rdna of Freshwater Copepods Neodiaptomus Species and Mesocyclops Species See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/313010364 Phylogenetic analysis of 18s rDNA of freshwater copepods neodiaptomus species and mesocyclops species Article in Journal of Advanced Zoology · December 2016 CITATION READS 1 202 5 authors, including: Sivakumar Kandasamy M.R Dhivya Shree Karpaga Vinayaga College of Engineering and Technology KLE Institute of Technology 27 PUBLICATIONS 80 CITATIONS 2 PUBLICATIONS 2 CITATIONS SEE PROFILE SEE PROFILE P. Muthupriya Kareem Altaff D.G.Vaishnav College AMET DEEMED TO BE UNIVERSITY 17 PUBLICATIONS 17 CITATIONS 66 PUBLICATIONS 350 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Meiofaunal studies of Palk bay sandy beaches View project Copepods View project All content following this page was uploaded by Sivakumar Kandasamy on 28 January 2017. The user has requested enhancement of the downloaded file. J. Adv. Zool. 2016: 37(2): 64-74 ISSN-0253-7214 PHYLOGENETIC ANALYSIS OF 18S rDNA OF FRESHWATER COPEPODS NEODIAPTOMUS SPECIES AND MESOCYCLOPS SPECIES K. Sivakumar1, K. Archana1, M. Shree Rama1, P. Muthupriya2 and K. Altaff3 1Department of Biotechnology Karpaga Vinayaga College of Engineering and Technology GST Road, Chinna Kolambakkam, Padalam-603 308, Kanchipuram (Dt.), India 2Department of Biotechnology DG Vaishnav College, Chennai – 600 106 3Department of Zoology The New College, Chennai-600 014 †corresponding author: [email protected] ABSTRACT: The present work is emphasized on analyzing the molecular characteristics of Mesocyclops sp. and Neodiaptomus sp. Molecular markers 18s rDNA region was used to resolve the evolutionary relationship between the species. The Mesocyclops sp. and Neodiaptomus sp. were sequenced and deposited in Gen Bank database. The similarity sequence was searched by BLAST programme and data was retrieved for construction of phylogenetic tree among the group. The percentage of similarity was found in the range of 80-83% and 97-100% in 18s rDNA region of Neodiaptomus sp. and Mesocyclops sp. to other species respectively. The phylogenetic tree was constructed using Neighbour-Joining method. Neodiaptomus sp. 18s rDNA region shows close relationship to Leptodiaptomus siciloides, Eudiaptomus wolterecki and Mastigodiaptomus albuquerquensis. Mesocyclops sp. 18srDNA region shows high similarity to Thermocyclops crassus and Mesocyclops leuckarti and less similarity to Mesocyclops edax clone. KEYWORDS: Neodiaptomus, Mesocyclops, NJ method and BLAST INTRODUCTION nutritional quality of copepods is Zooplankton plays an important role generally accepted to be very good for in energy and matter transfer through the marine fish larvae, and belived to be of a aquatic ecosystem1. Most zooplankton higher quality than the commonly used occupies second or third trophic level of live food Artemia5,6. Cyclopoidae is the the aquatic food web. In zooplankton, mostly found family among copepod copepod and cladoceran are the dominant species. The joint lies between fourth and groups in freshwater2. Copepods form a fifth segments of the body7. Calanoida is subclass belonging to subphylum another important species in an order of crustaceans, and it divided into ten copepods, they include around 40 orders. But only three are common in families, with about 1800 species of both plankton samples: such as Cyclopoida, marine and freshwater copepod. Poecilostomatida and Calanoid3,4. The 64 J. Adv. Zool. 2016 : 37 (2) Morphological identification copepod biodiversity and it is effective methods provide accurate information for the study of species level about species diversity8. Microscopic identification of calanoid copepods17. 18s discrimination of copepods is feasible rRNA is widely used for diversity only at the late copepodite and adult research in eukaryotes and also it has stages9. Identification of copepods at egg been used for resolving phylogenetic and larval stages are difficult using relationship among cyclopoid copepods microscope and also this method is time at higher level18. Nuclear DNA consuming and ambiguous. Therefore, it approximately 166,000 times larger than is necessary to develop alternative mtDNA and also provides set of markers method for identification of diversity of that potentially segregate independently samples10. Molecular techniques have and also phylogenetic studies carried at potential to provide species identification higher systemic level19. A molecular even the smaller aquatic organisms10. phylogenetic study is to recover the order Molecular techniques are useful for evolutionary events and represent them in identification of the species at early stage evolutionary trees that graphically depict level and shown sometimes significant relationship among species or genus over genetic differentiation among species11,12. time20. In molecular level, different techniques MATERIALS AND METHODS are available such as RFLP, RAPD, Collection and Preservation of the Multiplex PCR and genomic DNA sample: sequences used to discriminate the The Copepods samples were closely related specie13. Recently DNA collected from Padalam, Kancheepuram sequencing provides thousands rather District using plankton net (0.35 mouth than tens of characters enabling the diameter) made up of bolting silk cloth reconstruction of better resolved and (No 10, 158µm for 20-30 mins). The more robust phylogenetic trees14. samples were transported to the Currently few nuclear and mitochondrial laboratory immediately and they were markers have been used to successfully fixed in 90% ethanol for taxonomical resolve phylogenetic relationship in studies. The copepod had identified on copepods15,16. The main reason for the basis of morphological by using a selecting these two markers is highly binocular microscope. Copepod samples conserved regions. are first screened through 500µm mesh to The nuclear rRNA is the good remove fish and prawn larvae. The molecular marker for the study of rinsing is made repeatedly to reduce the 65 J. Adv. Zool. 2016 : 37 (2) contamination after rinsing the desired Amplification conditions consisted of copepod is picked from zooplankton 5min at 950C and then 40sec at 950C, samples with the help of fine capillary 25sec at 500C, 3min at 720C for 40 tubes and needles. Then the copepod is cycles, and extension for 15min at 720C washed with double distilled water and 23. preserved in 90% ethanol to facilitate Gene Sequencing: DNA isolation 21. DNA sequencing was carried out on DNA Isolation: an automated DNA sequencer. Proof DNA was isolated by Saline reading, editing, and alignments were Citrate Solution method (SCS). 200mg of performed using the program Sequencer copepod samples were suspended in 4.1 24. 800µl of saline citrate solution (0.14M Phylogenetic Analysis: Nacl and 0.02M Tri Sodium Citrate) and DNA homology searches were homogenized by morter and pestle. The performed using BLASTn 2.2.24 homogenate is transferred into a fresh programs at NCBI and similarity centrifuge tube and centrifuged at sequences were retrieved for 3000rpm for 10min and after phylogenetic analysis. Obtained centrifugation the supernatant will be sequences were verified, corrected and discarded and the pellet is resuspend in assembled with MEGA tool and aligned Saline Citrate Solution and then by ClustalW algorithm. Phylogenetic centrifuged at 3000 rpm for 5min. Again relationships were evaluated of 18s rRNA the pellet resuspended in 400µl of 2M nuclear genes by Neighbour Joining Nacl solution and centrifuged at method 25. 10000rpm for 15min at 40C. Followed by centrifugation the supernatant was RESULTS AND DISCUSSION collected in fresh centrifuge tubes. To this DNA Isolation and amplification: double the volume of ethanol is added The DNA was extracted and and the DNA is allowed to precipitation amplified of 18s rDNA region from for 6min 22. Neodiaptomus sp. (Fig. 1) and DNA Amplification: Amplification Mesocyclops sp. (Fig. 2). The molecular reaction of the 18s rDNA gene will be weight of amplified DNA was 1176 carried out using the primers 18s rRNA (Neodiaptomus sp.) and 1363 bp Forward 5’-GCA AGT CTG GTG CCA (Mesocyclops sp.). DNA Isolation done GCA GCC -3’ and 18s rRNA Reverse 5’- by using SCS method extracted DNA in TGA TCC TTC CGC AGG TTC AC -3’. the range of 2027 to 23130 bp for both 66 J. Adv. Zool. 2016 : 37 (2) Neodiaptomus sp. and Mesocyclops sp. in Lane 1 – Lambda DNA / Hind III Digest 18s rDNA region. The extracted DNA Marker; Lane 2 – Genomic DNA. Lane 1 – 1Kb DNA Ladder; Lane 2 – PCR was further amplified and nucleotide product. sequence of 1176 and 1363 base pair of 18s rDNA gene sequence analysis 18s rDNA was determined for Neodiaptomus sp. (Fig 1) and The 18s rDNA gene is one of the Mesocyclops sp. (Fig 2), respectively. most frequently used molecular marker Fig.1: DNA Isolation and PCR product of 18s for eukaryotes in determining generic and rRNA region of Neodiaptomus species specific level relationships and higher order analyses of phylogeny26. Similarity of sequences of Neodiaptomus sp. and Mesocyclops sp. was retrieved by BLASTn program. List of accession numbers and organism are give below in Table 1 and 2. The Neodiaptomus sp. and Mesocyclops sp. found maximum identity of 98% and 100% for 18s rDNA region, respectively. Blastn program were used to find the similarity of species based on maximum identity and E value. In 18s Lane 1 – Lambda DNA / Hind III Digest Marker; Lane 2 – Genomic DNA. Lane 1 – rDNA region of Neodiaptomus species
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