http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–2 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1060455

MITOGENOME ANNOUNCEMENT

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839)

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Sudhanshu Raman1, A. Pavan-Kumar1, Prakash G. Koringa2, Namrata Patel2, Tejas Shah2, Rajeev K. Singh3, Gopal Krishna1, C. G. Joshi2, P. Gireesh-Babu1, Aparna Chaudhari1, and W. S. Lakra1 1

Division of Fish Genetics and Biotechnology, ICAR – Central Institute of Fisheries Education (Deemed University), Mumbai, India, 2Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India, and 3 ICAR – National Bureau of Fish Genetic Resources, Dilkusha PO, Lucknow, India Abstract

Keywords

The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. The genome sequence was 16 573 bp in size, and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A: 31.9%, G: 15.6%, C: 27.68%, T: 24.76%, A + T content 56.6% and the G + C content 43.32%. The phylogenetic tree constructed using a maximum likelihood model showed sister relationship between T. khudree and Tor tambroides.

Mitogenome, next generation sequencing, phylogeny

The Deccan mahseer, Tor khudree (Sykes, 1839) (Order: Cypriniformes, Family: Cyprinidae) is widely distributed in major rivers and reservoirs of central and peninsular India. However, because of overfishing and habitat destruction, the population size of this species has reduced drastically and it is now listed as an endangered species in the IUCN Red List (Raghavan, 2013; Raghavan et al., 2008). Further, information on the available genetic resources of mahseers in India is highly ambiguous and led to taxonomic instability (Khare et al., 2014). DNA-based taxonomy with large genomes (mitogenomes) would resolve taxonomic ambiguity better than single gene fragments. With this background, the present study was carried out to sequence the complete mitochondrial genome of endangered Deccan mahseer. Samples of T. khudree were collected from Periyar river, Kerala, India and fins were taken under aseptic conditions and preserved in absolute alcohol at 80  C for further processing. Total genomic DNA was isolated from preserved fin tissue following SDS-phenol/chloroform method described by Sambrook et al. (2001). The complete T. khudree mitochondrial genome was amplified into two overlapping amplicons (9 kb) using a long PCR technique (for primers and thermal conditions see Kim & Lee, 2004). The amplicons (500 ng) were sequenced using Ion torrent platform and a total of 277,157 sequence reads (average length 240 bp) were generated covering 66 Mb of mitogenome with coverage of 4000. The length of complete mitochondrial genome of T. khudree was 16,573 bp in size Correspondence: A. Pavan-Kumar, Scientist, Division of Fish Genetics and Biotechnology, Central Institute of Fisheries Education, Versova, Andheri West, Mumbai 400 061, India. Tel: +91 02226361447x452. E-mail: [email protected]

History Received 29 May 2015 Accepted 30 May 2015 Published online 8 July 2015

(GenBank Accession No. KR868706) with a overall base composition of 31.9% for A, 24.76% for T, 27.68% for C and 15.64% for G. The complete mitogenome of T. khudree includes 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding and a control region (D-loop). The ribosomal RNA, tRNA and protein coding genes of T. khudree were predicted and annotated using Mito annotator tool (Iwasaki et al., 2013). As reported in other fish species, in T. khudree also all proteincoding genes were encoded on H-strand (heavy strand) except ND6 protein coding gene. Few of the tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNAGlu and tRNA-Pro) were encoded on L-strand (light), while most of the other tRNA genes were present on H-strand. Except cytochrome c oxidase subunit I (start codon: GTG), all other protein coding genes share the start codon ATG. It was wellknown fact that many of the fishes use incomplete codons to stop the protein translation (Oh et al., 2007). In this species also, 7 of 13 protein coding genes are terminated with incomplete codons of T (CytB, ND2, ND3, ND4, COII, COIII) or TA (ATPase6). Remaining five protein coding genes (ND1, COI, ATPase 8, ND4L, ND5 and ND6) have TAA termination codon. Among all the protein coding genes, ND5 gene (1826 bp) is the longest one and the shortest gene is ATPase 6 (164 bp). The two ribosomal RNA genes, 12S rRNA gene (955 bp) and 16S rRNA gene (1674 bp) are separated by tRNA-Val and this entire gene cassette is flanked by tRNA-Phe and tRNA-Leu. The length of the control region (D-loop region) is 917 bp. Further, we validated the present study species status by investigating its phylogenetic position within Cyprinidae family. The phylogenetic tree constructed by a maximum likelihood method using complete mitochondrial genome of 13 species of Cyprinidae family revealed that T. khudree is unambiguously

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Mitochondrial DNA, Early Online: 1–2

Figure 1. Molecular phylogeny of T. khudree with other related species in Cyprinidae family based on the complete mitogenome. The complete mitogenomes were downloaded from GenBank and the phylogenic tree was constructed by a maximum likelihood method with 500 bootstrap replicates. The gene’s accession number for tree construction is listed as follows: Esomus metallicus (AB239594.1), Hypophthalmichthys nobilis (NC_010194.1), Hypophthalmichthys molitrix (NC_010156), Neolissochilus hexagonolepis (NC_026106.1), Tor sinensis (NC_022702), Tor tambroides (NC_023243), Tor putitora (Catla catla (NC_016892), Labeo rohita (NC_017608), Cirrhinus mrigala (NC_017611), Cyprinus carpio (NC_001606), Carassius auratus (NC_002079), T. khudree (present study species), Schizothorax labiatus (NC_023365).

grouped with other tor species with significant bootstrap value (Figure 1). In conclusion, in this study we sequenced the complete mitochondrial genome of endangered mahseer (T. khudree) using Ion torrent platform. This mitogenome information would be useful to resolve the taxonomic ambiguity exists in this group and to study the phylogeography of Tor species.

Declaration of interest The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References Iwasaki W, Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP, Sado T, et al. (2013). MitoFish and MitoAnnotator: A mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol Biol Evol 30:2531–40.

Khare P, Mohindra V, Barman SA, Singh KR, Lal KK. (2014). Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India. Organ Divers Evol 14:307–26. Kim IC, Lee JS. (2004). The complete mitochondrial genome of the rockfish Sebastes schlegeli (Scorpaeniformes, Scorpaenidae). Mol Cells 17:322–8. Oh DJ, Kim JY, Lee JA, Yoon WJ, Park SY, Jung YH. (2007). Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae). DNA Seq 18:295–301. Raghavan R. (2013). Tor khudree. The IUCN Red List of Threatened Species. Version 2014.3. Available at: www.iucnredlist.org (Accessed 29 May 2015) Raghavan R, Prasad G, Ali A, Pereira B. (2008). Fish fauna of River Chalakudy part of Western Ghats biodiversity hotspot (South India) – Patterns of distribution, threats and conservation needs. Biodivers Conserv 17:3119–31. Sambrook SJ, Russel DW, Janssen KA, Irwuin NJ. (2001). Molecular cloning, a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839).

The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. ...
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