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