ISSN Original Article. Nucleotide sequence of 16S rrna gene of Clarias gariepinus and its molecular phylogeny with other catfishes

Available online at http://www.urpjournals.com International Journal of Research in Fisheries and Aquaculture Universal Research Publications. All rights reserved ISSN 2277-7729 Original Article Nucleotide sequence of 16S rrna gene of Clarias gariepinus and its molecular phylogeny with other catfishes Azmiri Sultana 1, Rowshan Ara Begum 2, Hawa Jahan 3, Mohammad Shamimul Alam 4 and Reza Mohammad Shahjahan 5 1. MS Student, Laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka, E-mail: mridul7771@hotmail.com, Phone: 0195-4395053; 2. Professor, Laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka, E-mail: rowshanbegumdu@yahoo.com; 3. Research Assistant, HEQEP Project, Department of Zoology, University of Dhaka; 4. Associate Professor, Laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka, E-mail: shamimul.du@yahoo.com; 5. Professor, Laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka E-mail: reza.shahjahan56@gmail.com. Received 21 February 2015; accepted 13 March 2015 Abstract Clarias gariepinus commonly known as the North American catfish, belong to a diverse group of ray finned fish. They are of considerable commercial importance; many of the larger species are farmed or fished for food. Partial sequence of 16S rrna gene of the mitochondrial genome in the catfish Clarias gariepinus was determined that contained 561 base pairs (bp). Genomic DNA was isolated successfully by CTAB method. Amplification of DNA was carried out using universal set of primers which amplified partial region of the mitochondrial 16S rrna gene. Nucleotide sequence was determined using the Sanger dideoxy sequencing method. The sequence was 561 bp long and the A+T base composition of the gene was about 53%. Interspecific and intraspecific comparison between two catfishes namely C. gariepinus and C. batrachus based on the 16S rrna gene. The intraspecific sequences of 16S rrna gene of C. gariepinus was aligned and 8 of polymorphic sites were observed. However, 21 polymorphic sites were revealed after comparing the interspecific sequence between C. gariepinus and C. batrachus. These polymorphic sites could be used as a molecular tool for identification of these two catfishes. Furthermore, partial 16S rrna sequences of a few different locally found catfishes from Bangladesh and India were collected from the GenBank database, all of which were over 500 bp long. A phylogenic tree was constructed using the multiple sequence alignment data of the 8 catfish sequences to determine their phylogenic relationship. All eight catfishes form a monophylic group that belong to the order Siluriformes. Among different species of catfish C. gariepinus and C. batrachus form a monophylic group with high bootstrap value (98). Both of these fishes are consistent with phylogeny based on morphology and they belong to the family Clariidae. Labeo rohita and Channa striata were used as out groups. This research demonstrates that partial sequences of this gene can identify the different species of catfish, indicating the usefulness of mtdna-based approach in species identification. 2015 Universal Research Publications. All rights reserved Key words: Sequence of 16S rrna gene, mtdna, catfish, C. gariepinus, C. batrachus, phylogeny. Introduction Mitochondria are structures within cells that convert the energy from food into a form that cells can use. Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. This genetic material is known as mitochondrial DNA or mtdna. In eukaryotes, mitochondrial DNA is a circular molecule spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 2 genes for ribosomal subunits, one for the smaller and the other for the larger subunit. The genes are knows 12S rrna and 16S rrna in that order. 16S rrna is the particular region of DNA that has proved to be the most helpful for evolutionary correlation between different species. The genes coding for 33

it are referred as 16S rdna and are used it reconstructing phylogenies (Woese, 1977). 16S ribosomal RNA is a component of the 30S small subunit of prokaryotic ribosome and is 1.5 kb in length. Although it was originally used to identify an organism, 16S sequencing was subsequently found to be capable of reclassifying the organism into completely new species, or even genera (Weisburg et al., 1991). 16S rrna has several functions. Like the large (23S) ribosomal RNA of nuclear origin, it has a structural role, acting as a scaffold defining the positions of the ribosomal proteins. 16S rrna has a lot of other important economical uses. This gene along with other mitochondrial genes are used to ensure quality checks on canned fishes and meat and investigate the status of cancerous disease, toxicological status of fish. These mitochondrial genes are also used for the purposes of environmental study. 16S rrna gene sequence is also used in the bar coding of certain vertebrates, for examples amphibian, fishes etc. It also serves as a molecular phylogenetic tool in key invertebrate species such as Drosophila. Comparative performance of 16S rrna gene in certain amphibians was investigated (Vences et al., 2005). In this study experimental evidence was provided that mitochondrial 16S rrna gene fulfills the requirement for a universal DNA barcoding marker in amphibians. Phylogenetic position of the African lungfish in Sudan was inferred from the complete sequence of the 12S rrna and 16S rrna genes (Abukashawa, S. & Daghestani, M.). This study demonstrated that the 12S rrna and 16S rrna alignment results showed that members of the 2 lungfish subspecies are sister groups and the coelacanth are more closely related to the tetrapods than the lungfish of Sudan Phylogenetic relationships among all members of D. virilis Sturtevant species group were concluded from partial 16S rrna and 12S rrna sequence data in the molecular phylogenetic study performed by S.G Spicer and C.D. Bell (2002). Li Yang (2014) worked on the species identification through mitochondrial rrna genetic analysis. Inter-species and intraspecific variations in mitochondrial DNA (mtdna) were observed in a bioinformatics analysis of the mitochondrial genomic sequences of 11 animal species. The molecular phylogenetic approach using the mtdna has been extensively conducted in fish because it is a valuable marker for detecting genetic differences at the species level and for providing high-resolution information on the evolutionary relationships between taxonomically close families (Santos et al., 2003). Especially, the 16S rrna gene is reasonably well conserved and have been sequenced in various invertebrate and vertebrate taxa (Moller and Gravlund, 2003; Ann et al, 2005). No previous study has been previously performed on the mitochondrial genome of C. gariepinus in Bangladesh. C. gariepinus also known as the North African catfish are elongated fishes with fairly long dorsal and anal fins. The dorsal fin has 61-80 soft rays and the anal fin has 45-65 soft rays. They have strong pectoral fins with spines that are serrated on the outer side. It belongs to the order Siluriformes and family Clariidae along with another member Clarias batrachus. These two are the only locally found species belonging to the Genus Clarias (Rahman, Ataur A.K. Freshwater Fishes of Bangladesh). The members of Clariidae family are scaleless, snake headed thick skinned fishes. Along with them there are other families belonging to this particular order with different morphological features, belonging to different families. Heteropneustes fossilis and H. microps belong to the family Heteropneustidae, they have a long slender scaleless body with a bony head. Ompok pabda belongs to family Siluridae; members of this family have an elongated compressed head with a pair of maxillary and mandibular barbels and a short dorsal spine. In this study we determined a partial sequence of the 16S rrna of C. gariepinus. The sequence was aligned with sequences of catfishes from Bangladesh and India and they were compared. The multiple sequence alignment allowed us to study the number of polymorphic sites and henceforth the phylogenetic relationships between these fishes based on the 16S rrna sequence of C. gariepinus was investigated. Materials and Methods Sample Collection: The African sharptooth catfish or locally known as the African Magur (Clarias gariepinus) was collected from a local market in Azimpur, Dhaka. The fresh specimen was brought to the laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka for further experiment. 3 fresh Eppendorf tubes were taken and two muscle samples were collected from the dorsal part of the body of the specimen. The samples were loaded in two of the tubes and named as CG1 and CG2, and the other one was kept as a negative control. DNA extraction: Total genomic DNA was extracted by phenol: chloroform extraction method (Begum et al. 2004b) with slight modification. The fresh specimens were dissected after measuring length and weight (50 cm; 500g) to collect the required tissue sample. Samples were squashed in 500 µl CTAB buffer. 10 µl Proteinase K was added and the sample mixture was inverted to mix properly. An equal volume of Phenol: Chloroform was added to the sample mixture. The mixture was spun again at 13,000 rpm at room temperature for 5 minutes and lower Phenol-Chloroform phase was removed. DNA is found at the upper aqueous phase. Sample was precipitated with 1 volume of 100% Ethanol at room temperature and inverted several times. DNA was pelleted by centrifugation at 13000 rpm for 5 minutes and washed with 70% alcohol, dried and dissolved 50 µl sterilized distilled water for 10 minutes. The extracted DNA was then visualized in 1.0% agarose gel of a low melting point grade (Life Technologies, USA) at 120 V for 20 minutes. The extracted DNA samples were added to wells constructed in the gel, along with a loading dye (Bromophenol Blue) in a 5:1 ratio. DNA amplification: DNA fragments encoding the 16S rrna gene were amplified by Polymerase Chain Reaction. The chemicals used for the amplification of mitochondrial DNA were 2 µl of 10X PCR Reaction Buffer, 1.6 µl of 25 mm MgCl 2, 0.5 µl of 10 mm dntp, 0.5 µl of 16S rrna forward primer (Primer- F), 0.5 µl of 16S rrna reverse primer (Primer- R), 0.16 µl of Taq Polymerase, 2 µl 34

Template DNA, and sterilized distilled water is used to make up a total volume of 25 µl. Amplifying conditions were 93 C for 60s in denaturation, 53 C for 90s in annealing, and 72 C for 90s in extension for 30 cycles with a final polymerization step at 72 C for 5 minutes. PCR products were resolved by electrophoresis in a 1.5% agarose gel of a low melting point grade (Life Technologies, USA), amplified DNA samples were added to wells constructed in the gel, along with a loading dye (Bromophenol Blue) in a 5:1 ratio and stained with Ethidium Bromide to visualize DNA bands. Dilution of the template DNA is required because the isolated DNA was highly concentrated and unsuitable for using in PCR. DNA Template was diluted with sterilized distilled water in a ratio of 1:9. For the dilution of the primer equal volume of sterilized distilled water was added to the tube as its molecular weight. The primers used in the amplification of the DNA fragments are as follows, forward primer Primer- F (5 -CGC CTG TTT AAC AAA AAC AT-3 ) and reverse primer Primer- R (5 -CCG GTT TGA ACT CAG ATC ATG T-3 ). The PCR products of interest were then purified using Favor Prep PCR Clean up Mini Kit (Favorgen Biotech Corp.) and then washed in 70% ethanol dried and re-suspended in 50 µl of sterilized distilled water. DNA Sequencing: After the amplified DNA was purified successfully, its sequence was determined by Sanger Dideoxy Sequencing method using (enter name of sequencer) in Centre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka. Sequence Collection from NCBI: Partial sequences of mitochondrial genome of various locally found catfishes and catfishes from a different geographical location in this case India, were collected from NCBI. The collected samples and their respective accession numbers are as follows, Clarias gariepinus Bangladesh (submission number: 1725601), C.gariepinus India 1 (JQ699187), C. gariepinus India 2 (JQ699187), C. batrachus Bangladesh (KF997532), Clarias batrachus India 1 (GQ402540), Clarias batrachus India 2 (JQ699193), Heteropneustes fossilis (KJ819943), H. microps (FJ432686) Pangasius pangasius (GQ411088), Ompok bimaculatus (GQ469642), O. pabda (GQ469569), Wallago attu (KJ911222). Partial 16S rrna of Labeo rohita (KC757195) and Channa striata (HM117250) were also collected and used as an out group. All the sequences were over 500 bp in length. The sequences were aligned and phylogenetic tree was constructed depending on the multiple sequence alignment using the software SeaView for a colorful output and also to visualize sequence similarity and dissimilarities easily. Result and Discussion DNA Extraction: DNA extraction of both the samples were performed successfully. Extracted DNA was visualized in 1% agarose gel. Bands of isolated DNA observed visualized under the transilluminator, were clear and concise as shown in Figure 1. Figure 1. 1.0% Agarose gel showing the bands of isolated DNA of samples from the collected specimen Clarias gariepinus as denoted by CG1 and CG2. DNA Amplification: Partial region of 16S rrna gene of C. gariepinus amplified by PCR method using universal primers Primer-F (5 -CGC CTG TTT AAC AAA AAC AT-3 ) and Primer-R (5 -CCG GTT TGA ACT CAG ATC ATG T-3 ). The amplified DNA of these samples were then observed by running them through Agarose gel of concentration 1.5% (Figure 2). Compared with DNA marker it was revealed that the amplified gene was over 500 bp long. The sequence can be found on the GenBank database and its submission number is 1725601. Figure 2. 1.5% Agarose gel showing the bands of amplified DNA of 16S rrna of C. gariepinus (denoted by CG1 and CG2) and N denotes negative control. The fourth lane shows the DNA marker. 35

Sequences of Clarias gariepinus: Altogether 561 bp of nucleotide sequence was determined. The sequence can be found on the GenBank database and its submission number is 1725601. Figure 2 shows the schematic position of the amplified partial region of the 16S rrna gene. To compare with other C. gariepinus samples sequence data files containing various lengths of partial sequences of 16S rrna of C. gariepinus was collected from NCBI which were all over 500 bp long. The collected data contained partial sequences of 16S rrna submitted by researchers from National Bureau of Fish Genetic Resources, India. The collected data were then arranged carefully along with the sequenced sample of the present study. Figure 3. A schematic diagram of a complete 16S rrna gene of C. gariepinus, where the shaded portion shows the amplified region and sites of oligonucleotides used as primers (arrows) for PCR. from Bangladesh and India. The intraspecific alignment Intraspecific variation of sequences: The current study between sequence samples of other catfishes such as was performed to investigate the intraspecific comparison Clarias batrachus, Heteropneustes fossilis collected from between C. gariepinus from Bangladesh and India based on Bangladesh and India shows a polymorphic site percentage the 16S rrna gene. The percentage of polymorphic sites of 0.004% for C. batrachus (2 polymorphic sites out of 517 found between intraspecific alignments of the specimen nucleotide bp). Figure 5 and Table 2 includes the used in current study and the sequences collected from the intraspecific alignment pattern and the respective GenBank database belonging to specimen found in India polymorphic sites found between 3 C. batrachus sequence are 0.014% (8 polymorphic sites out of 561 nucleotide base samples. The percentage of polymorphic site found in the pairs). The results shows that this gene is pretty much sequences of Heteropneustes fossilis from Bangladesh and conserved in between species. Figure 4 and Table 1 shows India is 0.03% (15 out of 553 nucleotide base pairs; data the intraspecific alignment pattern and the number of not shown). polymorphic sites found between C. gariepinus samples Figure 4. Intraspecific alignment pattern between samples of C. gariepinus from Bangladesh and India. 36

Interspecific variation of sequences: Interspecific comparison can classically be defined as the comparison of gene sequences between different organisms of the same genus to identify the regions of similarities and dissimilarities. In this study the interspecific variations of partial regions of 16S rrna gene between C. gariepinus and C. batrachus from Bangladesh and India was also studied. The 16S rrna sequence data files for C. batrachus from both locations was collected from NCBI and a comparative study was performed. The sequences were aligned using Seaview and 21 polymorphic sites were observed (Figure 4 and Table 2). A total of 517 base pairs between 6 sequence samples of the interspecies was compared. The percentage of polymorphic site is 0.04%. Table 3 shows the base composition and the percentage of polymorphic sites found in the interspecific comparison between different catfishes from Bangladesh and India. The detailed interspecific comparison data for catfishes apart from the ones in belonging to the genus Clarias are not shown. Table 2. Polymorphic sites observed in between C. gariepinus and C. batrachus species from Bangladesh and India. Sequences labelled CG stands for C. gariepinus and CB stands for C. batrachus, Ind 1 and 2 and Bang represents their respective locations which are Bangladesh and India. The - sign indicates deletion of a nucleotide base pair. Table 3. Base composition and the percentage of polymorphic sites found in the interspecific comparison between different catfishes from Bangladesh and India. Figure 5. Interspecific alignment pattern between samples of C. gariepinus and C. batrachus from Bangladesh and India. Accession numbers: Clarias gariepinus Bangladesh (present study), C.gariepinus India 1 (JQ699187), C. gariepinus India 2 (JQ699187), C. batrachus Bangladesh (KF997532), Clarias batrachus India 1 (GQ402540), Clarias batrachus India 2 (JQ699193), Heteropneustes fossilis (KJ819943), H. microps (FJ432686), Ompok bimaculatus (GQ469642), O. pabda (GQ469569). 37

Phylogenetic analysis: A comprehensive study was performed among catfishes using the neighbor joining method which falls under the category of distance method of phylogenetic investigation. Siluriformes is clearly form a monophylic group when Labeo rohita and Channa striata was used as an out group. Monophyly of the catfishes in the present study agreed with the classification reported by Alves Gomes & Lundberg (1993). Labeo rohita (KC757195) and Channa striata (HM117250) was included as an out group. Therefore from the data it can be observed that 16S rrna shows a more conserved result amongst closely related individuals. C. gariepinus and C. batrachus, belonging to the family Clariidae form a monophylic group with a bootstrap value of 98. H. fossilis and H. microps belongs to the family Heteropneustidae forms a monophylic group with a bootstrap value of 90 and it shows closest relation to C. gariepinus and C. batrachus having a bootstrap value of 93. If we consider the morphological classification, Heteropneustidae is closest to Clariidae due to similarity in morphological features such as thick bony head, scaleless skin, streamlined body and the number of barbels. Apart from these aforementioned families Ompok pabda and O. bimaculatus belonging to the Family Siluridae also forms a monophylic group with a bootstrap value of 74. Apart from the high bootstrap values all eight catfishes are consistent with phylogeny based on morphology, and therefore to their respective families. Figure 5 shows the phylogenetic relationship between the C. gariepinus sequence used in the present study along with other locally found catfishes based on the neighbor joining distance method. Figure 6. Molecular phylogenic tree for Clarias gariepinus (Submission no: 1725601), C. batrachus (KF997532) Heteropneustes fossilis (KJ819943), H. microps (FJ432686), Ompok bimaculatus (GQ469642), O. pabda (GQ469569), Pangasius pangasius (GQ411088), Wallago attu (KJ911222) according to the neighbor joining method based on the partial nucleotide sequences for the 16S rrna gene. Labeo rohita (KC757195) and Channa striata (HM117250) were used as an out group. Nucleotide sequences of all the species are from the GenBank database and were used to root the tree. Conclusion From the discussion above we can conclude that all catfishes in the present study form a monophylic group when based on 16S rrna gene sequence when Labeo rohita and Channa striata was used as an out group. 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