Sadequr Rahman Khan
Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh, Bangladesh
Habiba Akter
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Nargis Sultana
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Mohd. Golam Quader Khan
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Md. Abdul Wahab
Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh, Bangladesh
Md. Samsul Alam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Macrobrachium rosenbergii; Microsatellite; Heterozygosity; Genetic diversity
Bagerhat, Barguna and Cox’s Bazar district.
Conservation and Biodiversity
Pond and Hapa Preparation: Nine experimental ponds each with an area of 100 m2 (0.01 hectare) were prepared by draining out of water and removing weeds and predators. Agricultural lime (CaCO3) was applied at the rate of 250 kg ha-1 on the bottom of the pond and the pond was filled up with underground water using a deep tube well. For promoting algal growth, the ponds were fertilized with urea, Triple Super Phosphate (TSP) and cow dung at the rates of 25 kg ha-1, 25 kg ha-1 and 750 kg ha-1 respectively. Nine hapas, made of fine meshed nylon net, each having 10 m2 in area (5 m × 2 m) with 1 m height were set up by using bamboo poles maintaining the top of the net 1ft above the water level. Collection, Stocking and Management of Post-larvae (PLs) of M. rosenbergii: PLs were collected from three rivers namely the Pashur, the Paira and the Naaf under Bagerhat, Barguna and Cox’s Bazar district, respectively and transported to the Fisheries Field Laboratory Complex, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh. After conditioning for three days, the PLs with an average weight of 0.23 g were stocked at a rate of 3 PL m-2 (30 PL/hapa) with three replications. Supplementary feed having 32% protein was applied at 10% of total body weight up to 40 days and then the feeding ration was adjusted to 5% of the body weight for the next 30 days. The feed was applied twice a day, half in the morning and the other half in the afternoon. Collection of Tissue Sample and Extraction of Genomic DNA: After 70 days of rearing, the juvenile prawns from the three hapas under each river source were mixed. The mean individual harvest weights (g) of the juvenile prawn were 1.51, 0.90 and 1.30 g in the Pashur, Paira and Naaf population, respectively. The pleopods of 36 randomly collected samples from each of the three river sources were clipped by using scissors and then preserved in 95% ethanol and stored at -20oC. The genomic DNA was extracted from approximately 30 mg of the tissue following standard phenol-chlorofrom-isoamyl alcohol extraction and ethanol precipitation method. The quality of the DNA samples was checked by electrophoresis on 1% agarose gel and the quantity was measured using a spectrophotometer. PCR Amplification: Initially, ten pairs of microsatellite primers (Mbr1, Mbr2, Mbr3, Mbr4, Mbr5, Mbr7, Mbr8, Mbr9, Mbr10, Mbr11) from M. rosenbergii were screened but finally seven pairs were used for genetic diversity assessment of three river populations of the freshwater prawn. The PCR was conducted using 50 ng template DNA, 2.0 μM of each primer, 0.25 mM of each dNTPs, 1X PCR buffer and 1 unit of Taq DNA polymerase (GENEI, India) in a total volume of 12 μL. A gradient thermocycler (Eppendorf, Germany) was used to conduct the PCR under the following temperature profiles: an initial denaturation step at 94ºC followed by 35 cycles each consisting of denaturation at 94ºC for 30 sec, annealing at 56ºC for 30sec and extension at 72ºC for 1 min. A final step of elongation at 72ºC for 5 min followed the last cycle. For confirmation of PCR amplification, the products were mixed with 3 μL of loading dye (0.25% Bromophenol blue, 0.25% Xylene cyanol, 30% glycerol) and one half was run on 2% agarose gel. Polyacrylamide Gel Electrophoresis and Silver Nitrate Staining: For separation of the microsatellite alleles, the PCR products were electrophoresed on 6% denatured polyacrylamide gel (19:1:polyacrylamide:bisacrylamide) containing 7 M urea using Sequigen GT (Bio-Rad, Hercules, USA) vertical gel electrophoresis system. The silver nitrate staining protocol of Promega (Madison) was followed for staininig the polyacrylamide gel fixed on the glass plate. Statistical Analysis of Microsatellite Data: The length of the alleles against the molecular weight markers were estimated using the software Alpha Ease FC (Version 4.0). The genotype data on all loci of all samples of the three populations were compiled in a matrix using the Microsoft Excel program. Analysis of various population genetic parameters such as the number of alleles (Na), effective number of alleles (Ne), frequency of alleles, observed (Ho) and expected heterozygosity (He), Nei’s (1972) genetic distance and gene flow (Nm) between the population pairs and chi-square test for fit to Hardy- Weinberg Expectation (HWE) and Analysis of Molecular Variance (AMOVA) were performed using the software GenAlEx version 6.4. The software ARLEQUIN version 3.0 was used to test the statistical significance between the FST values of different population pairs applying 1000 permutations of the genotypes. The software MEGA version 4 was used to build a UPGMA (unweighted pair-group method of averages) dendrogram.
Int. J. Agric. Biol., Vol. 16, No. 1, 195-200, 2014
Journal