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Chromosomal Studies and Quantitative Karyotypic Analysis of Rohu, Labeo Rohita

Md. Sarower-E-Mahfuj
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh

M. Belal Hossain
Biology Group, Faculty of Science, Univesity Brunei Darussalam, BE 1410, Brunei

M. Mokhlesur Rahman
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh

Md. Imran Hoshan
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

Chromosomal and karyotypic studies are required for genetic improvement of any organisms. This study was performed to identify individual chromosomes on morphological basis and to characterize a standard karyotype using the fish, Rohu (Labeo rohita, Hamilton, 1822). Colchicine (0.05%) treated (2, 2.5 and 3 h) tissues of two day-old larvae were used for slide preparations and selected plates were photomicrographed under high resolution research microscope. Slide preparations were done following hydrolysis (10% HCl), mordanting (2% iron alum) and staining with haematoxylin. Colchicine treatment for 2 h gave satisfactory results in respect of degree of contraction of the chromosomes. Chromosome number 2n = 50 was counted at metaphase stage. Measurements of the chromosomes were taken from the selected plates based on morphologically distinct condition. Standard haploid karyotype was formulated following the combined scatter diagram technique. Six individually identifiable and 19 not individually identifiable chromosomes consisted of the haploid complement. The six individually identifiable ones consisted of 2 m+3 sm+1st and not individually identifiable ones consisted of 16 m+3 sm chromosomes. This study report may provide complete report on chromosomal and karyotype knowledge in L. rohita and suggest the genetic purity of L. rohita may contributes to sustainable aquaculture production.

Keywords: Karyotype, Chromosome, Haploid, Metaphase and colchicin

Location: Cytogenetics laboratory, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Carp fish

Objectives:

The present investigation aims to provide the detailed information on the chromosome number and quantitative karyotype of Rohu, Labeo rohita.

Methodology:

Sample collection and processing: Two day-old alive, larvae were collected from “Brahmaputra Fish Seed Complex”, Shambugonj, Mymensingh, Bangladesh. The larvae were transported to the Cytogenetics laboratory, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh. For chromosomal studies and quantitative karyotypic analysis method was followed with some modifications. The stock solution of colchicine was made by dissolving 0.05 g colchicine in 100 mL distilled water. Healthy and vigorously growing larvae were allowed to swim in 0.05% colchicine solution. Then the larvae were kept in a glass petri dish containing 0.7% NaCl for 15 min at 28°C room temperature. Then the larvae were transferred to another petri dish containing distilled water kept for 15 min. The larvae were transferred into a vial containing freshly prepared fixative solution (acetic acid: ethanol, 1:3). Slide preparation: Three batches of fixation, 2, 2.5 h, were processed separately. Fixed larvae tissues were hydrolyzed in small vials with 10% HCl at 60°C in an incubator for 2-3 min. Before actual hydrolysis of the tissues 10% HCl was kept in the incubator at 60°C for one hour in a tightly stoppered vial. Fixed tissues were then kept in the same preheated HCl solution in the incubator. Then hydrolized tissues were washed in fresh water for 2-3 times. The tissues were then treated with 2% aqueous solution of iron alum (Ferric ammonium sulphate) for about 7 min for mordanting. Iron alum treated tissues were then washed for 2-3 times with fresh water and stained in 0.5% haematoxylin for 8 min. The stained tissues were kept in water. Slides were prepared within 2-3 h of staining and microscopic observation. Observation: Then mitotic dividing cells were observed among the spreading tissues by using (Olympus microscope, CHD-64M061, Japan). Photomicrographs of the selected chromosome plates were taken with the aid of Olympus research microscope (Olympus-BX40, Japan) using plan 100 X oil immersion objective. Chromosome measurements in millimeters (mm) were made from photo prints and chromosome length were converted to micron (μ) based on calculated final print magnification. Statistical analyses: Arm ratios of the chromosomes were calculated by dividing the length of the long arm by that of the short arm (L/S). Scatter diagram using the arm ratio and total length of the individual chromosomes at each of the selected plates were required. Homologous pairs of chromosomes were determined based on the proximity of two points and haploid values of the chromosomes were calculated. Thus a standard karyotype of L. rohita was developed following three steps of analysis: Firstly, a scatter diagram was produced for all chromosomes in each plate separately, by use of which the diploid number of chromosomes were reduced to the haploid number and haploid values of the chromosomes were determined. Secondly, a combined scatter diagram of the haploid complements of all plates was constructed to establish a standard morphology of those chromosomes which could be identified. Thirdly, those chromosomes which could not be identified individually were characterized through probabilistic inferences. In a combined scatter diagram six groups of points could be delineated, representing six individually identifiable chromosomes. Individually identified groups were encircled by boundaries around those chromosomes. Each group of points included four haploid values, one point of each plate. The mean and standard deviation were measured in terms of total length and arm ratio of four haploid values of each plate of a group boundary. Thus a group boundary represented a haploid identified chromosome. Unidentified chromosomes were classified according to length and arm ratio. These unidentified chromosomes were distributed to the various morphological classes. These were determined by probabilistic inferences based on the frequency in a given class which were occurred in the combined scatter diagram. Finally, all chromosomes in the haploid complement were numbered in decreasing order of length and increasing order of arm ratio.

Source of Information: Pakistan Journal of Biological Sciences, 17: 490-496, ISSN 1028-8880

Article Link (Online Journal): DOI: 10.3923/pjbs.2014.490.496; URL: http://scialert.net/abstract/?doi=pjbs.2014.490.496

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Chromosomal and karyotype have become an active area of research in fish genetics. This study report may provide a complete report on chromosomal and karyotype knowledge in L. rohita and suggest the genetic purity of L. rohita which may contributes to sustainable aquaculture production. The quantitative method used in this study may be appropriate for other fish species which may present similar cytological difficulties and may encourage further work on the cytogenetics especially in cytology and karyotype analysis.

Knowledge Management: Journal