http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–2 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1038795
MITOGENOME ANNOUNCEMENT
Complete mitochondrial genome organization of Tor tor (Hamilton, 1822) Rohit Kumar, Chirag Goel, Prabhati Kumari Sahoo, Atul K. Singh, and Ashoktaru Barat
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/15 For personal use only.
Molecular Genetics Laboratory, Directorate of Coldwater Fisheries Research, Nainital, Uttarakhand, India
Abstract
Keywords
The complete mitochondrial genome of Tor tor, a threatened ‘‘Mahseer’’ was sequenced for the first time. The mitochondrial genome size determined to be 16,554 bp in length and consisted of 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNA genes and a control region or displacement loop (D-Loop) region, resembling the typical organizational pattern of most of the teleost. The overall base composition found was A: 31.8%, T: 25%, G: 15.7% and C: 27.4%; A + T: 56.9% and G + C: 43.1%. The phylogenetic tree constructed using 11 other cyprinids’ total mtDNA datasets confirmed the location of present species among mahseers. The total sequence data could support further study in molecular systematics, species identification, evolutionary and conservation genetics.
Genetic characterization, phylogeny, Tor mahseer
Tor tor (Fam:Cyprinidae), commonly known as ‘‘Mahseer’’, is an important fish for food and sport in Himalayan rivers. The species is ranked as ‘‘near threatened’’ (IUCN, 2014). All the mahseer species were characterized morphologically as well as using molecular markers (Nguyen et al., 2008; Sati et al., 2013), but certain level of taxonomic ambiguities do persist among them (Laskar et al., 2013). Tor tor was initially described as Cyprinus tor (Hamilton, 1822) and then characterized under different genera, namely Barbus, Puntius and Tor (Desai, 2003). In the present study, authors sequenced complete mitochondrial sequence of T. tor (16,554 bp; KP795444) and compared the sequence with the available mitogenomes of mahseer species to delineate its relation with con-generic species. Few reference mitogenomes from genera Cyprinus, Barbus, Punctius, Neolissochilus and Tor were also analyzed to depict the taxonomic position of Tor tor. Specimens of T. tor were collected from River Sharda, Uttarakhand, India (80 070 20 00 E, 29 050 0700 N) and were morphologically identified using the key characters given by Talwar & Jhingran (1991). Voucher specimen of the same was deposited to the museum (corresponding address) with ID TTSU-01. Complete mitochondrial genome was amplified as contiguous, overlapping fragments of 17 primer pairs (Cheng et al., 2012; Mabuchi et al., 2006; Wang et al., 2000). The PCR amplicons were sequenced using ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The contigs were checked for quality, assembled and annotated using CLC Genomics Workbench v.7.5.2 and reference databases (Schattner et al., 2005). All the gene arrangements, initiation and stop codons and length of coding sequences
History Received 3 March 2015 Revised 30 March 2015 Accepted 5 April 2015 Published online 28 May 2015
resembles to the closest congeneric species, Tor putitora (Sati et al., 2014). The mitogenome also contains two noncoding regions (Zhang et al., 2014): (1) putative replication origin of L-strand (OL) (34 bp, 5305–5338 bp) and (2) a 906 bp control region. When PCGs were compared to 3 other mahseer species, the lowest mean similarity was found in ND1 gene (92 %) and it might be considered suitable for characterization of mahseer species. Higher mean similarity were found in other genes that are used for species identification, namely Cyb (94.5%) and CO1 (96.6%). The reference mitogenomes of four different generas Cyprinus, Barbus, Puntius and Tor were retrieved from NCBI and used in phylogenetic analysis. Phylogenetic tree was constructed using concatenated nucleotide sequences from 12 PCGs (Figure 1) as well as ND1 gene (similar topology, data not shown). After the best fitted model test, General Time reversible model (+G, +T) was implemented in ML method to infer phylogeny followed by selection of topology with superior log likelihood value (47526.35). The tree topology clearly suggests the heterospecificity of Tor tor from all other mahseer species used in present study. The phylogeny also suggests the common ancestral origin of mahseer species, i.e. Tor and Neolissochilus but their relation to the Naziritor could not be inferred due to the unavailability of any reference sequence. Finally, the study supports the classification of Tor mahseer under genus Tor as earlier reported (Nguyen et al., 2008; Sati et al., 2013). The authors strongly recommend the use of complete mitogenome or ND1 alone for phylogenetic studies and molecular characterization of mahseer species.
Correspondence: Dr Ashoktaru Barat, Ph.D, Molecular Genetics Laboratory, Directorate of Coldwater Fisheries Research, Anusandhan Bhawan, Industrial Area, Bhimtal, Nainital 263136, Uttarakhand, India. E-mail: [email protected]
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/15 For personal use only.
2
R. Kumar et al.
Mitochondrial DNA, Early Online: 1–2
Figure 1. Phylogenetic tree (1000 replicates) constructed using 12 concatenated protein coding genes encoded on H-strand.
Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
References Cheng Y-Z, Xu T-J, Jin X-X, Tang D, Wei T, Sun Y-Y, Meng F-Q, et al. (2012). Universal primers for amplification of the complete mitochondrial control region in marine fish species. Mol Biol 46:727–30. Desai VR. (2003). Synopsis of biological data on the tor mahseer Tor tor (Hamilton, 1822). FAO Fisheries Synopsis. No. 158. Rome, FAO. 36p. [Online] Available at: http://www.fao.org/3/a-y4642e.pdf (Accessed 25 February 2015). IUCN. (2014). The IUCN red list of threatened species. Version 2014.3. [Online] Available at: www.iucnredlist.org (Accessed 25 February 2015). Laskar BA, Bhattacharjee MJ, Dhar B, Mahadani P, Kundu S, Ghosh SK. (2013). The species Dilemma of Northeast Indian mahseer (Actinopterygii: Cyprinidae): DNA barcoding in clarifying the riddle. PLoS One 8: e53704. Mabuchi K, Miya M, Senou H, Suzuki T, Nishida M. (2006). Complete mitochondrial DNA sequence of the Lake Biwa wild strain of common
carp (Cyprinus carpio L.): Further evidence for an ancient origin. Aquaculture 257:68–77. Nguyen TTT, Na-Nakorn U, Sukmanomon S, ZiMing C. (2008). A study on phylogeny and biogeography of mahseer species (Pisces: Cyprinidae) using sequences of three mitochondrial DNA gene regions. Mol Phylogenet Evol 48:1223–31. Sati J, Goel C, Kumar R, Ali S, Patiyal RS, Singh VK, Sahoo PK, Barat A. (2014). Complete mitochondrial genome organization of Tor putitora. Mitochondrial DNA 25:278–9. Sati J, Sah S, Pandey H, Ali S, Sahoo PK, Pande V, Barat A. (2013). Phylogenetic relationship and molecular identification of five Indian Mahseer species using COI sequence. J Environ Biol 34:933–9. Schattner P, Brooks AN, Lowe TM. (2005). The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686–9. Talwar PK, Jhingran AG. (1991). Inland fishes of India and adjacent countries, Vol 1. New Delhi, India: Oxford & IBH Pub. Co. Wang H-Y, Tsai M-P, Tu M-C, Lee S-C. (2000). Universal primers for amplification of the complete mitochondrial 12S rRNA gene in vertebrates. Zool Stud 39:61–6. Zhang X, Song W, Wang Y, Du R, Wang W. (2014). The complete nucleotide sequence of white Amur bream (Parabramis pekinensis) mitochondrial genome. Mitochondrial DNA 25:361–2.
MITOGENOME ANNOUNCEMENT
Complete mitochondrial genome organization of Tor tor (Hamilton, 1822) Rohit Kumar, Chirag Goel, Prabhati Kumari Sahoo, Atul K. Singh, and Ashoktaru Barat
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/15 For personal use only.
Molecular Genetics Laboratory, Directorate of Coldwater Fisheries Research, Nainital, Uttarakhand, India
Abstract
Keywords
The complete mitochondrial genome of Tor tor, a threatened ‘‘Mahseer’’ was sequenced for the first time. The mitochondrial genome size determined to be 16,554 bp in length and consisted of 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNA genes and a control region or displacement loop (D-Loop) region, resembling the typical organizational pattern of most of the teleost. The overall base composition found was A: 31.8%, T: 25%, G: 15.7% and C: 27.4%; A + T: 56.9% and G + C: 43.1%. The phylogenetic tree constructed using 11 other cyprinids’ total mtDNA datasets confirmed the location of present species among mahseers. The total sequence data could support further study in molecular systematics, species identification, evolutionary and conservation genetics.
Genetic characterization, phylogeny, Tor mahseer
Tor tor (Fam:Cyprinidae), commonly known as ‘‘Mahseer’’, is an important fish for food and sport in Himalayan rivers. The species is ranked as ‘‘near threatened’’ (IUCN, 2014). All the mahseer species were characterized morphologically as well as using molecular markers (Nguyen et al., 2008; Sati et al., 2013), but certain level of taxonomic ambiguities do persist among them (Laskar et al., 2013). Tor tor was initially described as Cyprinus tor (Hamilton, 1822) and then characterized under different genera, namely Barbus, Puntius and Tor (Desai, 2003). In the present study, authors sequenced complete mitochondrial sequence of T. tor (16,554 bp; KP795444) and compared the sequence with the available mitogenomes of mahseer species to delineate its relation with con-generic species. Few reference mitogenomes from genera Cyprinus, Barbus, Punctius, Neolissochilus and Tor were also analyzed to depict the taxonomic position of Tor tor. Specimens of T. tor were collected from River Sharda, Uttarakhand, India (80 070 20 00 E, 29 050 0700 N) and were morphologically identified using the key characters given by Talwar & Jhingran (1991). Voucher specimen of the same was deposited to the museum (corresponding address) with ID TTSU-01. Complete mitochondrial genome was amplified as contiguous, overlapping fragments of 17 primer pairs (Cheng et al., 2012; Mabuchi et al., 2006; Wang et al., 2000). The PCR amplicons were sequenced using ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The contigs were checked for quality, assembled and annotated using CLC Genomics Workbench v.7.5.2 and reference databases (Schattner et al., 2005). All the gene arrangements, initiation and stop codons and length of coding sequences
History Received 3 March 2015 Revised 30 March 2015 Accepted 5 April 2015 Published online 28 May 2015
resembles to the closest congeneric species, Tor putitora (Sati et al., 2014). The mitogenome also contains two noncoding regions (Zhang et al., 2014): (1) putative replication origin of L-strand (OL) (34 bp, 5305–5338 bp) and (2) a 906 bp control region. When PCGs were compared to 3 other mahseer species, the lowest mean similarity was found in ND1 gene (92 %) and it might be considered suitable for characterization of mahseer species. Higher mean similarity were found in other genes that are used for species identification, namely Cyb (94.5%) and CO1 (96.6%). The reference mitogenomes of four different generas Cyprinus, Barbus, Puntius and Tor were retrieved from NCBI and used in phylogenetic analysis. Phylogenetic tree was constructed using concatenated nucleotide sequences from 12 PCGs (Figure 1) as well as ND1 gene (similar topology, data not shown). After the best fitted model test, General Time reversible model (+G, +T) was implemented in ML method to infer phylogeny followed by selection of topology with superior log likelihood value (47526.35). The tree topology clearly suggests the heterospecificity of Tor tor from all other mahseer species used in present study. The phylogeny also suggests the common ancestral origin of mahseer species, i.e. Tor and Neolissochilus but their relation to the Naziritor could not be inferred due to the unavailability of any reference sequence. Finally, the study supports the classification of Tor mahseer under genus Tor as earlier reported (Nguyen et al., 2008; Sati et al., 2013). The authors strongly recommend the use of complete mitogenome or ND1 alone for phylogenetic studies and molecular characterization of mahseer species.
Correspondence: Dr Ashoktaru Barat, Ph.D, Molecular Genetics Laboratory, Directorate of Coldwater Fisheries Research, Anusandhan Bhawan, Industrial Area, Bhimtal, Nainital 263136, Uttarakhand, India. E-mail: [email protected]
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/15 For personal use only.
2
R. Kumar et al.
Mitochondrial DNA, Early Online: 1–2
Figure 1. Phylogenetic tree (1000 replicates) constructed using 12 concatenated protein coding genes encoded on H-strand.
Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
References Cheng Y-Z, Xu T-J, Jin X-X, Tang D, Wei T, Sun Y-Y, Meng F-Q, et al. (2012). Universal primers for amplification of the complete mitochondrial control region in marine fish species. Mol Biol 46:727–30. Desai VR. (2003). Synopsis of biological data on the tor mahseer Tor tor (Hamilton, 1822). FAO Fisheries Synopsis. No. 158. Rome, FAO. 36p. [Online] Available at: http://www.fao.org/3/a-y4642e.pdf (Accessed 25 February 2015). IUCN. (2014). The IUCN red list of threatened species. Version 2014.3. [Online] Available at: www.iucnredlist.org (Accessed 25 February 2015). Laskar BA, Bhattacharjee MJ, Dhar B, Mahadani P, Kundu S, Ghosh SK. (2013). The species Dilemma of Northeast Indian mahseer (Actinopterygii: Cyprinidae): DNA barcoding in clarifying the riddle. PLoS One 8: e53704. Mabuchi K, Miya M, Senou H, Suzuki T, Nishida M. (2006). Complete mitochondrial DNA sequence of the Lake Biwa wild strain of common
carp (Cyprinus carpio L.): Further evidence for an ancient origin. Aquaculture 257:68–77. Nguyen TTT, Na-Nakorn U, Sukmanomon S, ZiMing C. (2008). A study on phylogeny and biogeography of mahseer species (Pisces: Cyprinidae) using sequences of three mitochondrial DNA gene regions. Mol Phylogenet Evol 48:1223–31. Sati J, Goel C, Kumar R, Ali S, Patiyal RS, Singh VK, Sahoo PK, Barat A. (2014). Complete mitochondrial genome organization of Tor putitora. Mitochondrial DNA 25:278–9. Sati J, Sah S, Pandey H, Ali S, Sahoo PK, Pande V, Barat A. (2013). Phylogenetic relationship and molecular identification of five Indian Mahseer species using COI sequence. J Environ Biol 34:933–9. Schattner P, Brooks AN, Lowe TM. (2005). The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686–9. Talwar PK, Jhingran AG. (1991). Inland fishes of India and adjacent countries, Vol 1. New Delhi, India: Oxford & IBH Pub. Co. Wang H-Y, Tsai M-P, Tu M-C, Lee S-C. (2000). Universal primers for amplification of the complete mitochondrial 12S rRNA gene in vertebrates. Zool Stud 39:61–6. Zhang X, Song W, Wang Y, Du R, Wang W. (2014). The complete nucleotide sequence of white Amur bream (Parabramis pekinensis) mitochondrial genome. Mitochondrial DNA 25:361–2.
