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Genetic Variation in Endangered Butter Catfish, Ompok Bimaculatus (bloch) Populations Revealed by Random Amplified Polymorphic DNA (rapd) Fingerprinting

Jonaira Rashid
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Samsul Alam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mostafa Ali Reza Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Faozia Mehbuba Tamanna
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

This study was conducted to provide baseline information on the genetic population structure of Ompok bimaculatus, an endangered catfish of Bangladesh. Random amplified polymorphic DNA (RAPD) fingerprinting analysis was performed to assess the genetic variation in two wild (Chalan beel and Tola haor) and one hatchery (Brahmmaputra Fish Seed Complex-BFSC) populations of O. bimaculatus,. Five selected decamer random primers amplified a total of 34 RAPD bands among which 24 were found to be polymorphic. The percentage of polymorphic loci, intra-population similarity indices, gene diversity and Shannon’s information indidex values were 64.71%, 77.57% , 0.249±0.216 and 0.365±0.303 for Chalan beel, 58.82%, 75.45%, 0.219±0.215 and 0.322±0.304 for Tola haor population and 52.94%, 86.49%, 0.214±0.219 and 0.311±0.312 for the hatchery, BFSC, respectively. The coefficient of population differentiation (PhiPT) between the Chalan beel - BFSC and Tola haor - BFSC pairs were found to be significant. The gene flow (Nm) between the population pairs ranged from 1.899 to 5.052. The highest inter-population similarity (Sij) was found between Chalan beel-BFSC populations. Among the three populations, the highest genetic distance (0.157) was found between Tola haor and the BFSC population. The results of the present study indicated a substantial level of genetic variation in the endangered O. bimaculatus populations in Bangladesh and significant differentiation among the populations..

Keywords: RAPD, Ompok bimaculatus, Endangered, Polymorphic loci.

Location: Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Start Date: 00-00-2009

End Date: 00-00-2009

Program Area: Variety and Species

Commodity: Cat fish

Objectives:

1. To provide baseline information on the genetic population structure of Ompok bimaculatus, an endangered catfish of Bangladesh.

Methodology:

Collection of fish sample and extraction of DNA: In order to study genetic variations within and among different populations of O. bimaculatus, samples were collected from three different sources: Chalan beel under Natore district and the Tola haor under Netrokona district during July-August, 2009. The hatchery samples were collected from Brahmmaputra Fish Seed Complex (BFSC), Mymensingh in September, 2009. The hatchery operator collected O. bimaculatus broods from a renowned natural water body of Bangladesh named as “Tanguar haor” in Sunamgonj district. In order to perform RAPD fingerprinting, a total 60 fish, 20 from each population were taken randomly. The fish were anesthetized with MS222 and the tissue samples were clipped from the caudal fin of each fish and immediately preserved in individual microfuge tube containing 95% ethanol and stored at -20°C. For isolation of genomic DNA, approximately 40 mg of fin tissues from each sample was cut into small pieces and ground with a tissue grinder in 1.5 ml microfuge tube. The genomic DNA was isolated following cell rupture and proteinase-K digestion, phenol: chloroform: isoamyl alcohol (25:24:1; v/v/v) extraction, and ethanol precipitation method. The quality of all DNA samples were tested (degradation of DNA) by electrophoresis on 1% agarose gel and the quantity was estimated using a photometer

(Biophotometer plus, Eppendorf, Germany). Polymerase chain reaction (PCR) and electrophoresis: We screened fifteen decamer primers from three kits (six from kit A, even from kit B and two from kit C) of random sequence (Operon technologies, Inc., Alameda, CA, USA) on random samples from each population. A final subset of five primers which displayed reproducible, scorable and clear bands was considered for analysis of the entire samples of 60 fish. PCR reactions were performed on each DNA in a 10 μl reaction mix containing 1 μl of 10× Taq polymerase buffer, 0.2μl of primer, 1 μl of dNTPs and 1 unit of Taq DNA polymerase (GENEI, Bangalore, India) and 150 ng μl of genomic DNA. The PCR was conducted using a gradient thermal cycler (Mastercycler Gradient Eppendorf, Germany). The reaction mix was preheated at 94ºC for 3 min followed by 40 cycles of 30 sec denaturation at 94ºC, 1 min annealing at 38ºC and 2 min DNA chain elongation or extension at 72ºC. After the last cycle, a final step of 7 min at 72ºC was added to allow complete extension of all amplified fragments followed by holding at 4ºC. The amplified products from each sample were separated electrophoretically on 1.4% ethidium bromide containing agarose gel (GENEI, India) and the image was captured using a Gel Documentation system. Collection and analysis of RAPD data: The image profiles of banding patterns were recorded and molecular weight of each band was determined by AlphaEaseFC (Version 4.0) software. The banding pattern was scored on the basis of presence or absence of clear, visible and reproducible bands. The RAPD fragments were compared among the samples collected from different populations. The data were analyzed based on the principle that a band is considered to be polymorphic if it is present in some individuals and absent in others. A single data matrix was constructed pooling the scores obtained for all primers in all individuals under the three populations and used to estimate gene frequency, polymorphic loci, gene diversity, observed number of alleles (N_a), effective number of alleles (N_e), Shannon’s Information Index (I), gene flow (N_m), genetic distance and homogeneity test (χ² test) between the population pairs using the POPGENE (Version 1.31) computer program. We examined the hierarchy of molecular variance and differentiation (Phi-PT) between the population pairs by AMOVA using the software GenAlex 6.4. The similarity index values (SI) between the RAPD profiles of any two individuals on the gels were calculated from RAPD markers of the same molecular weight on the data matrix.

Source of Information: International Journal of Biosciences (IJB) Vol. 2, No. 9, p. 85-93, 2012

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

A substantial level of genetic variation in the endangered O. bimaculatus populations in Bangladesh and significant differentiation among the populations..

Knowledge Management: Journal