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Differentiation of Clarias Batrachus, C. Gariepinus and Heteropneustes Fossilis by Pcr-sequencing of Mitochondrial 16s Rrna Gene

Mohammad Shamimul Alam
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

Hawa Jahan
Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh.

Rowshan Ara Begum
Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh.

Reza M. Shahjahan
Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh.

Abstract/ Executive Summary:

Heteropneustes fossilis, Clarias batrachus and C. gariepinus are three major catfishes of ecological and economic importance. Identification of these fish species becomes a problem when the usual external morphological features of the fish are lost or removed, such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCR- sequencing provides accurate alternative means of identification of individuals at species level. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCR amplification and compared with the same gene sequences available from other geographical regions. Multiple sequence alignment of the 16S rRNA gene fragments of the catfish species has revealed polymorphic sites which can be used to differentiate these three species from one another and will provide valuable insight in choosing appropriate restriction enzymes for PCR-RFLP based identification in future.

Keywords: PCR-sequencing, 16S rRNA, Heteropneustes, Clarias, Species identification

Location: Local fish markets (Dhaka, Bangladesh).

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cat fish

Objectives:

(1) To compare the 16S rRNA gene sequences of three catfishes from Bangladesh with the existing sequences of those in the GenBank database.

(2) To compare sequences among the three selected catfishes.

(3) To explore the possibility of identifying these three species using PCR-RFLP on 16S rRNA gene sequence.

Methodology:

Sample Collection and DNA Extraction: H. fossilis (locally known as Shing), C. batrachus (Magur) and C. gariepinus (African Magur) were collected from local fish markets (Dhaka, Bangladesh). DNA was extracted from 50 mg muscle tissue of each fish species following a standard method (Doyle and Doyle 1990) with modifications. CT AB extraction buffer (1M 10 ml Tris-HCl [pH 8], 0.5M 4ml Sodium-EDT A [pH 8], 8.18 g NaCl, 2g CTAB, dH₂O) and 10 µl of 20 mg/mL proteinase K were added with minced meat sample. The mixture was incubated at 55°C for 2 h. After digestion, the samples were centrifuged at 13000 rpm for 5 min and equal volume of Phenol: Chloroform was added to the supernatant. Then, DNA was precipitated with ethanol and dissolved in distilled water. PCR amplification and Sequencing: Universal primers for PCR of the 16S rRNA gene, 16Sar-L (5'-CCGGTCTGAAAAAAACAT) and 16Sar-H (5'-CCGGTCTGAA CTCAG ATCACGT) have been utilized. Standard PCR was performed using PCR Master Mix (Promega). Temperature regimes for PCR amplifications were as follows - initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30s, 55°C for 30s and 72°C on the last cycle. PCR products were run in an agarose gel containing ethidium bromide. The electrophoresis was performed at 90 V for 30 min and DNA bands were visualized on a UV transilluminator and photographed. Then, PCR amplicons were purified using a purification kit (FavorGen) and sequenced. Bioinformatics: l6S rRNA gene sequences of experimental fish species and existing sequences of related fish species were collected from GenBank database (NCBI). Suitable portion of each sequence was taken for further observation. A multiple sequence alignment was performed using ClustalW software to observe the polymorphic sites present among the sequences. Restriction endo-nuclease cutting sites were searched in the sequences of the l6S rRNA fragment analyzed further. 'Serial Cloner' software was used to observe restriction sites.

Source of Information: J. Asiat. Soc. Bangladesh, Sci. 41 (1): 51-58, June 2015, 1016-6947

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In a study of eight pecoran species, 16S rRNA gene was found to have a larger number of species-specific polymorphic sites compared to the cytochrome b gene indicating the usefulness of 16S rRNA gene for species identification. In this circumstance, analyzing partial sequences of 16S rRNA gene for molecular differentiation of three selected catfishes of Bangladesh is a timely work, if not late. The results also prove that how this work can help to differentiate these three species. Moreover, using appropriate restriction enzymes (as indicated by this study) these three species can be separated by RFLP as well, which will be a less time consuming and cheaper practice.

Knowledge Management: Journal