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Polymorphic Loci Analysis of 16s Ribosomal Rna Gene of Bombay Duck (harpadon Nehereus)

Sristy Saha
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Zannatul Ferdous
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Hawa Jahan
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Ashfaqul Muid Khandaker
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Reza Md. Shahjahan
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Rowshan Ara Begum
Genetics and Molecular Biology Laboratory, Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Attempts were made to combine morphological and molecular techniques for identification and analysis of evolutionary relationship of commercially important marine lizardfish (Bombay duck) from the Bay of Bengal. For molecular identification, mitochondrial DNA (mtDNA) was extracted using CTAB extraction protocol and 16S rRNA gene was amplified by PCR method. Morphological identification was supported by molecular data generated by 99% identity of BLAST search result of Harpadon nehereus. The nucleotide sequence of H. nehereus was 586 bp long and the G+C content was 51% and intraspecies polymorphic sites were 0.5%. According to interspecies polymorphic sites analysis, 4.9% polymorphism was observed when H. nehereus and H. microchir were compared. Phylogenetic tree was constructed and both the Harpadon spp. formed a monophyletic clade. This analysis will be used to study molecular diversity as well as conservation of this fish species.

Keywords: 16S rRNA gene, Polymorphic loci, mt-DNA, Harpadon nehereus, Loitta

Location: Department of Zoology, University of Dhaka

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Loitta

Objectives:

To investigate the genetic characteristics of H. nehereus in this study a partial sequence of the 16S rRNA gene of H. nehereus were determined. The sequence was aligned with other sequence of lizardfish to study the number of polymorphic sites and the phylogenetic relationships

Methodology:

The Bombay duck or locally known as loitta (Harpadon nehereus) was collected from Cox’s Bazar (Fishery Ghats, fish markets). The fresh specimen was brought to the laboratory of Genetics and Molecular Biology, Department of Zoology, University of Dhaka for further experiment. The sample was then morphologically identified by conventional method. The measurement of different body part of collected fish was recorded. The sample was stored in refrigerator, minimum at 4°C. DNA extraction, PCR amplification and sequencing: Genomic DNA was extracted from 0.05 gm muscle tissue of the observed fish species following a method (Doyle and Doyle 1990) with modification. 100 µl CTAB extraction buffer and 10 µl of proteinase k were added. The mixture was incubated at 56°C overnight. After digestion equal volume of phenol chloroform was added with the supernatant and centrifuged at 13,000 rpm for 5 min. DNA was precipitated with ethanol and dissolved in distilled water. Standard PCR was performed using universal primers and were analyzed by electrophoresis in a 1.5 % agarose gel (Faucher et al. 2016). Then, PCR amplification was purified using a purification Kit and sequenced using direct sequencing method. Bioinformatics analysis: 16S rRNA gene sequence of experimental fish species and other existing sequence of lizard fish were collected from Gene Bank database (NCBI). Local alignment was done with the help of serial cloner software. MEGA 7 software was used for multiple sequence alignment which was followed by polymorphic sites analysis and for construction of phylogenetic tree. Sequence alignment and pairwise distance calculation is used to conduct two or more biological sequences of similar length

Source of Information: Bangladesh J. Zool. 47(1): 49-57, 2019 ISSN: 0304-9027 (print) 2408-8455 (online)

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

As Bombay duck is a lizardfish, a comparative study was performed among lizardfish from Harpadontidae and synodontidae family using the neighbor joining method, with bootstrap value 1000 where Labeo rohita was used as an out group. In the present study, Harpadon nehereus and H. microchir, belong to the family Harpadontidae, form a monophyletic clade with bootstrap value 96. Monophyly of these two species of Harpadon in the present study agreed with the result based on cytochrome c oxidase sub unit I (COI) gene. Furthermore, Saurida wanieso and S. umeyoshii along with S. undosquamis belong to the family Synodontidae form a monophyletic clade with bootstrap value 99 and similar result was also observed based on COI gene. Where Harpadon nehereus was used in the present study and H. microchir (AP002919.1), S. wanieso (NC 025940.1), S. undosquamis (NC 003162.1), S. umeyoshii (KM189365.1) were collected from GenBank database. Labeo rohita (KR185963.1) was used as out group. Partial nucleotide sequence of 16S rRNA gene of Harpadon spp. of the present study was initially used to analyze the polymorphic loci and to construct a molecular phylogenetic tree. To confirm the present observation, further study is required based on nucleotide sequences of 12S rRNA, COI, cytochrome b, ND4 and ND1 genes.

Knowledge Management: Journal