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High Levels of Genetic Variability and Differentiation in Hilsa Shad, Tenualosa Ilisha (clupeidae, Clupeiformes) Populations Revealed by Pcr-rflp Analysis of the Mitochondrial DNA D-loop Region

Sabuj Kanti Mazumder
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh, Bangladesh.

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

Abstract/ Executive Summary:

The hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) is an important anadromous clupeid species from the Western division of the Indo-Pacific region. It constitutes the largest single fishable species in Bangladesh. Information on genetic variability and population structure is very important for both management and conservation purposes. Past reports on the population structure of T. ilisha involving morphometric, allozyme and RAPD analyses are contradictory. We examined genetic variability and divergence in two riverine (the Jamuna and the Meghna), two estuarine (Kuakata and Sundarbans) and one marine (Cox’s Bazar) populations of T. ilisha by applying PCR-RFLP analysis of the mtDNA D-loop region. The amplified PCR products were restricted with four restriction enzymes namely, XbaI, EcoRI, EcoRV, and HaeIII. High levels of haplotype and gene diversity within and significant differentiations among, populations of T. ilisha were observed in this study. Significant FST values indicated differentiation among the river, estuary and marine populations. The UPGMA dendrogram based on genetic distance resulted in two major clusters, although, these were subsequently divided into three, corresponding to the riverine, estuarine and marine populations. The study underlines the usefulness of RFLP of mtDNA D-loop region as molecular markers, and detected at least two differentiated populations of T. ilisha in Bangladesh waters.

Keywords: Genetic variability, PCR-RFLP, mtDNA, Tenualosa ilisha.

Location: Faculty of Fisheries, BAU.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Hilsa

Objectives:

1. To examine the usefulness of mitochondrial D-loop region diversity to supplement allozyme and RAPD data on the population genetic structure of T. ilisha.

Methodology:

Fish samples and extraction of DNA: Ninety hilsa (T. ilisha), with an average body weight of about 400 g, were collected from five geographic locales: Balashi (Gaibandha, upstream of the Jamuna River)), Chandpur (middle of the Meghna River), Kuakata An oil-free thermal cycler (Master Cycler Gradient Eppendorf, Germany) was used for PCR amplification with the following cycle parameters: initial denaturation at 94 °C for 3.0 min, followed by 35 cycles of ?denaturation at 94 °C for 45 s, annealing at 45 °C for 45 s and extension at 72 °C for 2.0 min. A final extension step at 72 °C for 7 min followed the last cycle. The PCR products were confirmed by electrophoresis on agarose gel and subjected to digestion with restriction endonucleases. RFLP analysis: Four restriction enzymes (XbaI, EcoRI, EcoRV and HaeIII) were used for digesting the amplified fragments. Ten microlitres of the PCR product were used for each digestion following manufacturer’s (Promega) recommended conditions. The restricted PCR products were electrophoresed on 1.4% agarose gel containing ethidium bromide and photographed under UV light using a Geldoc camera (UVP Inc). Two molecular weight markers, - DNA-HindIII digest and 100 bp DNA ladder were run on each gel along with the digested PCR products. The sizes of mtDNA restriction fragments were measured. Genetic data analysis and dendrogram construction: Restriction patterns generated from each restriction endonuclease were given letter designations in the order of their frequencies. Haplotype A refers to the most common digestion pattern in the analyzed samples. The remaining alphabetical profile names (B, C, etc.) indicate variant digestion patterns reflecting their frequencies in order. Composite haplotypes were constructed from all the enzymes used and arranged from the enzyme generating the fewest polymorphic patterns to that generating the most (XbaI, EcoRI, EcoRV and HaeIII in this order). The presence or absence of restriction sites in the control region was inferred for each of the four enzymes from a series of restriction fragment patterns. The site codes across the amplified region for a restriction enzyme were concatenated and each fish was assigned a code of four letters that described its composite, multi-enzyme haplotype. A single data matrix comprising the composite haplotypes and their frequencies in the five populations was constructed. Haplotype and gene diversity, both calculated and a hierarchical analysis of population subdivision performed using the analysis of molecular variance with 1000 simulated samples. Significance was assessed in both cases by generating random samples (number of simulated samples: 1000) under the hypothesis of selective neutrality and population equilibrium, as implemented in ARLEQUIN. Significance levels of pair-wise FST, under the hypothesis of no differentiation between populations, were determined by means of 10000 permutations of haplotypes between populations. Genetic distance values between population-pairs were calculated from the F_ST values of the respective population-pair by using the formula: D (genetic distance) = -log(1 - F_ST). Distance data were used to draw up an unweighted pairgroup method of averages (UPGMA) dendrogram.

Source of Information: Genetics and Molecular Biology, 32, 1, 190-196 (2009)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

High levels of haplotype and gene diversity within and significant differentiations among, populations of T. ilisha were observed in this study. Significant FST values indicated differentiation among the river, estuary and marine populations. The UPGMA dendrogram based on genetic distance resulted in two major clusters, although, these were subsequently divided into three, corresponding to the riverine, estuarine and marine populations. The study underlines the usefulness of RFLP of mtDNA D-loop region as molecular markers, and detected at least two differentiated populations of T. ilisha in Bangladesh waters.

Knowledge Management: Journal