http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–2 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1033710
MITOGENOME ANNOUNCEMENT
Complete mitochondrial genome sequence of Heteropneustes fossilis obtained by paired end next generation sequencing Lakshman Sahoo1, Santosh Kumar2, Sofia P Das1, Siddhi Patnaik1, Amrita Bit1, Jitendra Kumar Sundaray1, P Jayasankar1, and Paramananda Das1 Fish Genetics and Biotechnology Division, ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India and 2Fish Conservation Division, ICAR-National Bureau of Fish Genetics Resources, Lucknow, Uttar Pradesh, India
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 06/08/15 For personal use only.
1
Abstract
Keywords
In the present study, the complete mitochondrial genome sequence of Heteropneustes fossilis is reported using massive parallel sequence technology. The complete mitogenome of H. fossilis is obtained by de novo assembly of paired end Illumina sequences using CLC Genomics Workbench version 7.0.4, which is 16,489 bp in length. It comprised of 13 protein- coding genes, 22 tRNAs, 2 rRNA genes and a putative control region along with the gene order and organization, being similar to most of vertebrates. The mitogenome in the present study has 99% similarity to the complete mitogneome sequence of H. Fossilis, as reported earlier. Phylogenetic analysis of Siluriformes depicted that Heteropneustids were closer to Clariids. The mitogenome sequence of H. fossilis contributes better understanding of population genetics, phylogenetics and evolution of Indian catfish species.
CLC Genomics Workbench, Heteropneustes fossilis, mitogenome, next generation sequencing
The Indian catfish Heteropneustes fossilis (Bloch, 1974) belonging to the family Heteropneustidae, is an important tropical food fish and heavily exploited for food and medicine. Its population is declining day by day due to over fishing and habitat loss. Therefore, development of genomic resources including complete mitochondrial genome is the need of the hour. In the present study, we represented the complete mitochondrial genome of H. fossilis, with GenBank accession number KP345880. Pectoral fin from a single specimen of H. fossilis collected from Bhubaneswar, Odisha, was sampled and total genomic DNA was extracted by standard phenol-chloroform extraction method (Sambrook & Russell, 2001). Genomic DNA was prepared in paired-end libraries, tagged and subjected to next generation sequencing (NGS) on the Illumina Next-Seq 500 system (Genotypic Technology Pvt. Ltd, Bengaluru, India). Raw read pre-processing and de novo assembly was performed using CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark). The assembled mitogenome was annotated by comparing with previously published complete mitochondrial genome of H. fossilis (Nakatani et al., 2011) as well as mitogenomes of related catfish (Waldbieser et al., 2003; Zhao et al., 2013). A phylogenetic tree of Siluriformes was constructed based on 17 representative complete mtDNA sequences. The complete mitogenome of Indian catfish is 16,489 bp (Accession No KP345880) in length and comprised of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and a putative
Correspondence: Dr. P. Das, Principal Scientist, Fish Genetics and Biotechnology Division, ICAR- Central Institute of Freshwater Aquaculture, Kausalyaganga, BBSR-751002, Odisha, India. Tel. +91674-2465446. Fax. +91-674-2465407. E-mail: [email protected]
History Received 6 February 2015 Accepted 22 March 2015 Published online 27 May 2015
control region. H. fossilis mtDNA gene organization was in accordance with other vertebrates. Majority of the genes were encoded on the H-strand except ND6 gene and eight tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAGlu and tRNAPro), which were encoded on L-strand. Heavy strand over all base composition was as follows: A: 32.14%, C: 26.91%, G: 14.89%, T: 26.05% and A+T content: 58.19%. In all the protein coding genes, normally ATG was used as the start codon except COI that used GTG. Equally, TAA and an incomplete stop codon T was used by the protein coding genes of H. fossilis. However, like other vertebrates and catfish mitogenome, the stop codon TAG was not used by H. fossilis. Twenty-one tRNA genes of H. fossilis can fold into a typical cloverleaf structure, except for tRNASer (AGY) that lacks a dihydrouridine arm (D-arm), identified by the online software tRNAscan SE 1.21 (Lowe & Eddy, 1997). Length of tRNA genes varied from 66–75 bp. The control region, a major non coding region is found to be 859 bp in length. Three conserved sequence blocks (CSBI: 593–655 bp, CSBII: 714–737 bp and CSBII: 760–782 bp) and a stem loop structure with two TACAT within 44–91 bp of control region were observed. The putative origin of L-strand replication was found in a cluster of WANCY region (tRNATrp-tRNAAlatRNAAsn-tRNACys-tRNATyr) in the intergenic region between tRNAAsn and tRNACys, which has potential to fold into a stable stem loop structure (Wang et al., 2008). There were 5 overlap (1 bp to 10 bp) and 6 intergenic spacer (2 bp to 31 bp) regions. Phylogenetic tree revealed Heteropneustids were closer to Clariids (Figure 1) (Diogo, 2003). The present study depicts how massive parallel-sequencing technology can obtain mtDNA genome sequences without sample processing or primer designing, thereby overcoming the limitations of conventional sequencing strategy.
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 06/08/15 For personal use only.
2
L. Sahoo et al.
Mitochondrial DNA, Early Online: 1–2
Figure 1. Phylogenetic tree of Siluriformes by taking 17 representative mitochondrial complete genome sequences.
Acknowledgments Authors are thankful to the Director, ICAR-CIFA for providing laboratory facilities.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The work presented here was financially supported by the Indian Council of Agricultural Research under outreach activity, and Department of Biotechnology, Govt. of India, New Delhi.
References Diogo R. (2003). Higher-level phylogeny of Siluriformes: An overview. Catfishes 1:353–84.
Lowe TM, Eddy SR. (1997). tRNAscan-SE’: A program for improved detection of transfer RNA genes in genomic sequence. Nucl Acids Res 25:955–64. Nakatani M, Miya M, Mabuchi K, Saitoh K, Nishida M. (2011). Evolutionary history of Otophysi (Teleostei), a major clade of the modern freshwater fishes: Pangaean origin and Mesozoic radiation. BMC Evol Biol 11:177. Sambrook J, Russel DW. (2001). Molecular Cloning: A laboratory manual. New York: CSH Laboratory Press. Waldbieser GC, Bilodeau AL, Nonneman DJ. (2003). Complete sequence and characterization of the channel catfish mitochondrial genome. DNA Seq 14:265–77. Wang CH, Chen Q, Lu GQ, Xu JWi, Yang QL, Li SF. (2008). Complete mitochondrialgenome of the grass carp (Ctenopharyngodon idella, Teleostei): Insight into its phylogenic position within Cyprinidae. Gene 424:96–101. Zhao H, Kong X, Zhou C. (2013). The mitogenome of Pangasius sutchi (Teleostei, Siluriformes: Pangasiidae). Mitochondrial DNA 25:342–4.
MITOGENOME ANNOUNCEMENT
Complete mitochondrial genome sequence of Heteropneustes fossilis obtained by paired end next generation sequencing Lakshman Sahoo1, Santosh Kumar2, Sofia P Das1, Siddhi Patnaik1, Amrita Bit1, Jitendra Kumar Sundaray1, P Jayasankar1, and Paramananda Das1 Fish Genetics and Biotechnology Division, ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India and 2Fish Conservation Division, ICAR-National Bureau of Fish Genetics Resources, Lucknow, Uttar Pradesh, India
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 06/08/15 For personal use only.
1
Abstract
Keywords
In the present study, the complete mitochondrial genome sequence of Heteropneustes fossilis is reported using massive parallel sequence technology. The complete mitogenome of H. fossilis is obtained by de novo assembly of paired end Illumina sequences using CLC Genomics Workbench version 7.0.4, which is 16,489 bp in length. It comprised of 13 protein- coding genes, 22 tRNAs, 2 rRNA genes and a putative control region along with the gene order and organization, being similar to most of vertebrates. The mitogenome in the present study has 99% similarity to the complete mitogneome sequence of H. Fossilis, as reported earlier. Phylogenetic analysis of Siluriformes depicted that Heteropneustids were closer to Clariids. The mitogenome sequence of H. fossilis contributes better understanding of population genetics, phylogenetics and evolution of Indian catfish species.
CLC Genomics Workbench, Heteropneustes fossilis, mitogenome, next generation sequencing
The Indian catfish Heteropneustes fossilis (Bloch, 1974) belonging to the family Heteropneustidae, is an important tropical food fish and heavily exploited for food and medicine. Its population is declining day by day due to over fishing and habitat loss. Therefore, development of genomic resources including complete mitochondrial genome is the need of the hour. In the present study, we represented the complete mitochondrial genome of H. fossilis, with GenBank accession number KP345880. Pectoral fin from a single specimen of H. fossilis collected from Bhubaneswar, Odisha, was sampled and total genomic DNA was extracted by standard phenol-chloroform extraction method (Sambrook & Russell, 2001). Genomic DNA was prepared in paired-end libraries, tagged and subjected to next generation sequencing (NGS) on the Illumina Next-Seq 500 system (Genotypic Technology Pvt. Ltd, Bengaluru, India). Raw read pre-processing and de novo assembly was performed using CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark). The assembled mitogenome was annotated by comparing with previously published complete mitochondrial genome of H. fossilis (Nakatani et al., 2011) as well as mitogenomes of related catfish (Waldbieser et al., 2003; Zhao et al., 2013). A phylogenetic tree of Siluriformes was constructed based on 17 representative complete mtDNA sequences. The complete mitogenome of Indian catfish is 16,489 bp (Accession No KP345880) in length and comprised of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and a putative
Correspondence: Dr. P. Das, Principal Scientist, Fish Genetics and Biotechnology Division, ICAR- Central Institute of Freshwater Aquaculture, Kausalyaganga, BBSR-751002, Odisha, India. Tel. +91674-2465446. Fax. +91-674-2465407. E-mail: [email protected]
History Received 6 February 2015 Accepted 22 March 2015 Published online 27 May 2015
control region. H. fossilis mtDNA gene organization was in accordance with other vertebrates. Majority of the genes were encoded on the H-strand except ND6 gene and eight tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAGlu and tRNAPro), which were encoded on L-strand. Heavy strand over all base composition was as follows: A: 32.14%, C: 26.91%, G: 14.89%, T: 26.05% and A+T content: 58.19%. In all the protein coding genes, normally ATG was used as the start codon except COI that used GTG. Equally, TAA and an incomplete stop codon T was used by the protein coding genes of H. fossilis. However, like other vertebrates and catfish mitogenome, the stop codon TAG was not used by H. fossilis. Twenty-one tRNA genes of H. fossilis can fold into a typical cloverleaf structure, except for tRNASer (AGY) that lacks a dihydrouridine arm (D-arm), identified by the online software tRNAscan SE 1.21 (Lowe & Eddy, 1997). Length of tRNA genes varied from 66–75 bp. The control region, a major non coding region is found to be 859 bp in length. Three conserved sequence blocks (CSBI: 593–655 bp, CSBII: 714–737 bp and CSBII: 760–782 bp) and a stem loop structure with two TACAT within 44–91 bp of control region were observed. The putative origin of L-strand replication was found in a cluster of WANCY region (tRNATrp-tRNAAlatRNAAsn-tRNACys-tRNATyr) in the intergenic region between tRNAAsn and tRNACys, which has potential to fold into a stable stem loop structure (Wang et al., 2008). There were 5 overlap (1 bp to 10 bp) and 6 intergenic spacer (2 bp to 31 bp) regions. Phylogenetic tree revealed Heteropneustids were closer to Clariids (Figure 1) (Diogo, 2003). The present study depicts how massive parallel-sequencing technology can obtain mtDNA genome sequences without sample processing or primer designing, thereby overcoming the limitations of conventional sequencing strategy.
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 06/08/15 For personal use only.
2
L. Sahoo et al.
Mitochondrial DNA, Early Online: 1–2
Figure 1. Phylogenetic tree of Siluriformes by taking 17 representative mitochondrial complete genome sequences.
Acknowledgments Authors are thankful to the Director, ICAR-CIFA for providing laboratory facilities.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The work presented here was financially supported by the Indian Council of Agricultural Research under outreach activity, and Department of Biotechnology, Govt. of India, New Delhi.
References Diogo R. (2003). Higher-level phylogeny of Siluriformes: An overview. Catfishes 1:353–84.
Lowe TM, Eddy SR. (1997). tRNAscan-SE’: A program for improved detection of transfer RNA genes in genomic sequence. Nucl Acids Res 25:955–64. Nakatani M, Miya M, Mabuchi K, Saitoh K, Nishida M. (2011). Evolutionary history of Otophysi (Teleostei), a major clade of the modern freshwater fishes: Pangaean origin and Mesozoic radiation. BMC Evol Biol 11:177. Sambrook J, Russel DW. (2001). Molecular Cloning: A laboratory manual. New York: CSH Laboratory Press. Waldbieser GC, Bilodeau AL, Nonneman DJ. (2003). Complete sequence and characterization of the channel catfish mitochondrial genome. DNA Seq 14:265–77. Wang CH, Chen Q, Lu GQ, Xu JWi, Yang QL, Li SF. (2008). Complete mitochondrialgenome of the grass carp (Ctenopharyngodon idella, Teleostei): Insight into its phylogenic position within Cyprinidae. Gene 424:96–101. Zhao H, Kong X, Zhou C. (2013). The mitogenome of Pangasius sutchi (Teleostei, Siluriformes: Pangasiidae). Mitochondrial DNA 25:342–4.
