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Molecular Characterization of an Indigenous Minor Carp Bangana Dero

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Molecular Characterization of an Indigenous Minor Carp Bangana Dero Indian J. Anim. Res., 53(5) 2019: 594-598 AGRICULTURAL RESEARCH COMMUNICATION CENTRE Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com Molecular characterization of an indigenous minor carp Bangana dero (Cyprinidae: Labeoninae) of Manipur and its relationship with Labeonin fishes of North East India based on mitochondrial 16SrRNA gene sequences Ch. Basudha, N. Sobita*, A. Rawat and N. Prakash ICAR Research Complex for NEH Region Manipur Centre Lamphelpat, Imphal-795 004, Manipur, India. Received: 30-12-2017 Accepted: 01-06-2018 DOI: 10.18805/ijar.B-3554 ABSTRACT Bangana dero is an economically important indigenous cyprinid fish of Manipur, India having maximum standard length of 40cm. For the taxonomic placement of this species among the Labeonin fishes, molecular characterization is conducted by using mitochondrial 16SrRNA gene sequences. Partial sequences of mito. 16SrRNA gene of fifteen cyprinid species of the sub-family Labeoninae were downloaded from NCBI Genbank. The analysed nucleotide sequence lengths were 538 bp. In a total of 811 characters, 194 were conserved sites (monomorphic) and 347 were variable sites (polymorphic) and out of 347 variable sites, 73 were parsimony informative sites and 274 singleton sites. The nucleotide frequencies are 31.4% (A), 22.57% (T), 24.00% (C) and 21.96% (G). The transition/transversion bias (R) is 2.51. The overall mean distance is 0.083. The dendogram constructed by Neighbour-Joining tree have three clusters which can indicate the taxonomic positions of Bangana dero . Key words: Labeoninae, Mitochondrial 16SrRNA gene, Molecular phylogeny, Taxonomic relationship. INTRODUCTION unbranch and 17 branched. Caudal fin forked. Lateral line Bangana dero (Hamilton, 1822) common name is is complete. Lateral line scales 40-42. Scales are with red Kalabans known as Ngaton or khabak (in Manipuri), Khital marks and silvery in the belly (Talwar and Jingaran, 1991; (Tangkhul), Ngatai (Myanmar) is an endemic to Asia inland Vishwanth, 2002; Arunkumar, 2008). waters. Maximum standard length is 40cm. It is one of the Mitochondrial DNA sequences are frequently most popular indigenous minor carps in the North Eastern utilized for inferring phylogenetic relationship among states of India. It is distributed throughout the Himalayan organisms, because they have the properties of large copy foothills, in India, Nepal, China (Zhu, 1995) and Shri Lanka number, faster evolutionary rate, maternal inheritance, (Talwar and Jhingaran, 1991). It also found in Iran (Coad, smaller molecular weight and a lack of introns (Brown et 1995), Afghanistan (Petr, 1999) also found in Bangladesh al., 1979; Moritz et al., 1987). It has been proposed that (Rahman, 1989). The species is found in some of the national each species would be delineated by a particular sequence parks of Nepal, e.g., Koshi Tappu Wildlife Reserve, Chitwan or a tight cluster of very similar sequences. The mitochondrial National Park and Karnali National Park (Shreshtha, 1999). genome is the entirety of hereditary information. Sequence There is a need for improved habitat protection at sites where this species is known to occur. Further survey work is needed similarities serve as the proof for structural and functional to determine whether or not this species is experiencing a conservation. These markers generally exhibit 5 to 10 time decline, or is undergoing natural population fluctuations. greater variability than single copy nuclear genes, and have been used as a molecular tool for estimating phylogenetic This fish species can be distinguished from other relations in various groups of species. For the molecular cyprinids by the presence of small black blotch at base of phylogenetic study, mitochondrial 16S rRNA gene was used caudal fin and another above the pectoral fin. Body is in some fishes (Gilles et al., 1998, Li et al., 2013). These elongated, dorsal profile more curve than ventral. Head is genes are conserved and non-coding in nature which played moderate size, snout obtuse with pores and a small depression vital role in determination of new phylogenetic relationships across. Mouth is small, sub terminal. Lips are thick, papillated and in checking reliability of earlier established phyletic internally. There is one pair of barbells. Fins tinge red. Dorsal classification. fin inserted midway between tip of snout than to base of caudal fin and last quarter of pectorals with 3 unbranched The aim of present study is to establish the species and 10½ branched without spine. Pectoral fin with 1 identification as well as taxonomic placement of Bangana dero *Corresponding author’s e-mail: [email protected] Volume 53 Issue 5 (May 2019) 595 and its relationship with other fish species of the sub- RESULTS AND DISCUSSION family Labeoninae by using mitochondrial 16SrRNA gene The genomic DNA of all the collected samples were sequences. isolated and their gel photographs (0.8% Agarose) were MATERIALS AND METHODS Collection of fish samples: A total of thirty live fish Table 1: List of Labeonin fishes of North-East India with Genbank specimens of Bangana dero were collected from ICAR fish Accession Numbers. farms of ICAR Manipur Centre Lamphelpat, Imphal during Name of fish Genbank References the month of December, 2016. The morphological species Accession identifications were done according to the earlier literature Number cited (Vishwanath et al., 2014). For molecular identification, Bangana dero MG587929 Present study muscle tissue samples were collected and preserved in 70% Bangana dero MG595333 Present study alcohol until used. Bangana dero MG595334 Present study Bangana dero MG595335 Present study Isolation of genomic DNA: Genomic DNA was isolated by Bangana dero Mg595337 Present study using DNeasy Blood & Tissue Kit (Qiagen, Germany). Bangana dero MG595338 Present study PCR Amplification of mitochondrial 16S rRNA gene Bangana dero MG595340 Present study analysis: The mitochondrial 16S rRNA gene was amplified Bangana dero MG595341 Present study Bangana devdevi KT878057 NCBI using universal 16S rRNA primers (Palumbi, 1996). Thermal Cirrhinus cirrhosus JX074092 NCBI regime consisted of initial denaturation of 95°C for 5 mins., C. reba KC757165 NCBI followed by 35 cycles of denaturation (95 ºC for 40 sec), Garra annandalei JX074097 NCBI annealing for 30 sec at 45°C and extension 72 ºC for 40 sec G. gotyla JX074101 NCBI and post cycling extension 72ºC for 5 min and held at 4°C. G. gravelyi DQ845907 NCBI Electrophoresis was conducted on a 0.8% agarose gel at 100 G. kempi JX074086 NCBI V for 45 min to assess the success of DNA extraction and G. lissorhynchus KT878103 NCBI PCR amplified region. The gel was stained with ethidium G. litanensis KT878108 NCBI bromide prior to visualization for the presence of the G. nasuta JX074137 NCBI G. paralissorhynchus KT878114 NCBI extracted DNA, indicated by the presence of a band by using Labeo bata JX074105 NCBI 500 bp DNA step ladder as marker in a gel documentation L. dyocheilus JX074107 NCBI system (Gel Doc applied Biosystem, U.S.A.). L. gonius KT878177 NCBI Phylogenetic analysis: The products were sequenced L. rohita KJ425468 NCBI bidirectionally by using an automated ABI 3100 Genetic Pethia manipurensis KR909051 Sobita and Basudha, 2017 Analyzer (Eurofins, India). DNA sequences were edited by Bioedit and aligned by CLUSTAL X2. Alignment was then manually checked and corrected. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 5 (Tamura et al., 2011). Fifteen Labeonin fishes namely Bangana devdevi, Labeo bata, L. dyocheilus, L. gonius, L. rohita, Cirrhinus cirrhosus, C. reba, , Garra annandalei, G. gotyla, G. gravelyi, G. kempi, G. lissorhynchus, G. litanensis G. nasuta, and G. paralissorhynchus were downloaded from NCBI GenBank (Table 1). The phylogeny was established using the maximum parsimony (MP method). Fig 1: Gel photographs showing Genomic DNA isolated from the The evolutionary distances were calculated using the fish samples of Bangana dero. maximum composite likelihood method which was shown by the units of the number of base substitutions per site. All position containing gaps and missing data were eliminated from data set (complete deletion). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. One outgroup Pethia manipurensis was included in alignment as a root of tree. The split decomposition Fig 2: Gel photographs showing PCR amplified region of analysis were performed by using Splits Tree4 (version mitochondrial 16SrRNA gene (M- 500 bp DNA step ladder, 4:14:2) software for phylogenetic analysis of these labeonin Lane 1to 6-mitochondrial 16SrRNA gene region of fish fishes. species studied). 596 INDIAN JOURNAL OF ANIMAL RESEARCH shown in Fig 1. The isolated DNA were amplified in PCR (T), 24.00%(C) and 21.96%(G). The transition/transversion thermal cycler and size of the amplified sequences (540 bp) bias (R) is 2.51. The overall mean distance is 0.083. were confirmed by using 500bp DNA step ladder (Sigma, Phylogenetic analysis: The phylogenetic tree of Neighbor USA) by running in 1% gel (Fig. 2). The partial sequences Joining was constructed (Fig 3) from the combined dataset of mitochondrial 16SrRNA gene sequences for eight of mit. 16SrRNA sequences consisting of 23 sequences samples of Bangana dero were generated and deposited aligned with one outgroup as a root (Pethia manipurensis). in NCBI Genbank and their accession numbers are shown From dendrogram, three clusters were found. The first cluster in Table 1. consists of one genus i.e. Bangana with two species Bangana Nucleotide diversity: The analysed nucleotide sequence dero and B. devdevi. The second cluster is composed of two lengths were 538 bp. Here 811 characters were included out genera i.e. Cirrhinus and Labeo with six species namely of which 194 were conserved sites (monomorphic) and 347 Cirrhinus cirrhosus, C. reba, Labeo bata, L. dyochilus, L. were variable sites(polymorphic) and out of 347 variable gonius and L. rohita. The third cluster consists of one genera, sites, 73 were parsimony informative sites and 274 singleton Garra with eight species namely Garra annadalei, G.
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