Chromosomal Studies and Quantitative Karyotypic Analysis of Rohu, Labeo rohita
M. Belal Hossain,
M. Mokhlesur Rahman
Md. Imran Hoshan
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.
Received: April 10, 2013;
Accepted: May 08, 2013;
Published: November 26, 2013
Rohu, Labeo rohita is one of the important members of the family cyprinidae
which is distributed in native river systems of Bangladesh, India, Pakistan
and Myanmar (Talwar and Jhingran, 1991). In Bangladesh,
this species is mostly found in the Padma-Brahmaputra river systems and their
tributaries and in the Halda river of Chittagong region (Alam
et al., 2009). Rohu is a pioneer candidate among Indian Major Carps
which contribute 24.84% of the total fish production in Bangladesh (DoF,
2011). Recently the stock of Rohu population has been deteriorated through
uncontrolled interspecific and intergeneric hybridization and inbreeding (Sarder,
2007; Khan et al., 2006). As a result the
chromosomal and genetic constitution is altered resulting in deviation from
optimal growth and the purity of Rohu stocks is being gradually lost. It is
important to reveal the chromosomal and karyotypic condition in wild and hatchery
Rohu populations to identify pure and hybrid Rohu, to prevent loss of genetic
purity and protect them from potentially vulnerable to rapid deterioration.
Chromosomes are considered as the physical basis of heredity because they have
a special organization and are capable of self reproduction. From the viewpoint
of genetic control, the rapid production of inbred lines and cytotaxonomy, karyotypic
and chromosomal studies are important for fish breeding (Thorgaard
and Disney, 1990; Kirpichnikov, 1981). This type
of studies provide main information on the number, size and morphology of chromosomes
(Tan et al., 2004) which is needed to undertake
chromosome manipulation works (Khan et al., 2000).
In the last few decades works have been focused on the field of cytogenetic
investigation of fishes, especially in the area of systematics, mutagenesis
and aquaculture (Sofy et al., 2008).
In view of the above, L. rohita is a good candidate for genetic investigations
such as hybridization and ploidy manipulation. The present investigation aims
to provide the detailed information on the chromosome number and quantitative
karyotype of this species.
MATERIALS AND METHODS
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 the method of Ahmad
et al. (1983) 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). The conventions
proposed by Levan et al. (1964) were used to refer
to the different types of chromosomes. 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.
Clearly, three plates were found at late prophase stage, seven plates were
found in highly contracted condition of the chromosome and four plates were
found at prometaphase stage which were used in karyotypic studies. Colchicine
(0.05%) treatment for 2 h gave satisfactory results in respect of degree of
contraction of the chromosomes and frequency of prometaphase plate. The fixative
acetic acid and ethyl alcohol (1:3) was found suitable for the present materials.
Hydrolysis of the body tissues in 10% HCl for 2-3 min at 60°C was found
adequate to soften the specimen. Mordanting of the hydrolysed tissues for 7
min in 2% iron alum solution and staining for 8 min in 0.5% haematoxylin produced
adequate degree of staining of chromosomes. Present experiment revealed that
the somatic chromosome number of L. rohita was 2n = 50. Late prophase
stage was observed; the nuclear envelope breaks up and the chromosomes were
condensed. This is called super coiling (Fig. 1a). Exclusively
contracted metaphase chromosomes were also observed in the body tissues which
were unsuitable for measurement (Fig. 1b). A representative
plate of the four plates used in the karyotype analysis is shown in Fig.
Based on satisfactory staining and spreading four metaphase plates were selected
for karyotype analysis. From each of the single plate scatter diagram 25 homologous
chromosome pairs were determined by encircling the two proximal points (Fig.
||(a) Complement of two day-old larvae of L. rohita at late
prophase stage, showing thickly stained chromosome segments, (b) Chromosomes
highly contracted. Chromosomes at stages have shown 1 and 2 not used in
karyotypic analysis and (c) Representative complement of larvae tissue included
for karyotype analysis. All X 100
||Scatter diagram of a representative plate of L. rohita.
Each pair of points encircled was considered to represent a homologous pair
||Scatter diagram of the 25 haploid chromosomes values, lengths
and arm ratios, from each four plates of L. rohita; points A1-A25
for plate A; points B1-B25 for plate B; points C1-C25 for plate C and points
D1-D25 for plate D. Group boundaries were drawn around those chromosomes
which were recognizably homologous from each of the four plates. Intersecting
lines within the groups represent the group mean of total length and arm
ratio±one standard deviation for either variable of that group. Epithets
m1, sm1, st1 etc. within the groups are
the specific names of the chromosomes concerned
|| Mean lengths (μ) and mean arm ratios of the identified
chromosomes of L. rohita
The comparability of measurements of chromosomes in the four plates were determined
by examining the variation in haploid total lengths among the plates included.
The haploid total length ranged from 38.91-52.94 μ with a mean of 48.01±2.69
μ and a coefficient of variation of 5.60%. Thus, there was a similar degree
of contraction of the chromosomes in the four plates.
Corresponding chromosomes in different haploid complements were determined
through a grouping technique applied to the combined scatter diagram of the
four haploid compliments involving 100 haploid chromosomes shown in Fig.
3. Each point in the scatter diagram represented a specific chromosome in
a particular haploid complement. Symbols in the diagram refer to specific chromosomes
in a particular haploid complement and the adjacent letters A, B, C and D refers
to the four plates studied, respectively and numbers 1-25 represent the individual
haploid chromosomes. Thus one highest point on the Y axis, D25 represented the
haploid chromosome number 25 in plate D. Each cluster must contain one point
(chromosome) from each plate studied (Plates A-D).
Six groups of points could be recognized (Fig. 3). Each group
must contain four points belonging to four plates. These groups were clearly
distinct. The mean, standard error and coefficient of variation were determined
for length and arm ratio, using original diploid values. The six identified
chromosomes comprised 2 m, 3 sm and 1 st
types. The chromosomes within each type were numbered in decreasing order of
mean length. Chromosome type together with this number constitutes the specific
name of the chromosome concerned in Table 1. The six identified
chromosomes occupied approximately 28.61% of the total complement length and
this was consistent across four plates shown in Table 2.
||Locations of L. rohita chromosomes in a two-dimensional
array, based on total length and arm ratio. Solid circles represent identified
chromosomes, open circles represent unidentified chromosomes
|| Proportion of the complement total length occupied by the
six identified chromosomes in L. rohita
|| The allocation of unidentified chromosomes in L. rohita
karyotype to different morphological categories
All haploid chromosomes were classified according to length and arm ratio
shown in Table 3. Chromosome nomenclature was done based on
the categories of class interval of total length and arm ratio. This classification
was applied to the scatter diagram of the haploid complements as a grid of length
and arm ratio classes were shown in Fig. 4. Length was used
in plotting which resulted in vertical displacement of the points.
||Standard haploid karyotype of chromosomes with chromosome
The unidentified chromosomes were distributed to the various morphological
classes using probabilistic inferences based on the frequency of chromosomes
in a given class per haploid set in column 4 of Table 3, occurrence
of points in the combined scatter diagram in (Fig. 4) and
the original data and arm ratio of the chromosomes. The number of unidentified
chromosomes allocated to the various morphological classes is given in column
6 of Table 3. Finally, all 25 chromosomes in the haploid complement
were numbered from 1 to 25 in decreasing order of length and increasing order
of arm ratio within a length class shown in column 8 of Table
3. Thus each chromosome was allocated a serial identification number and
each of the six chromosomes identified individually also carried a specific
name based on its arm ratio. The morphological features of the chromosomes are
summarized in Table 4. The proposed standard karyotype consisted
of 18 m,
and 1 st
The chromosomes are considerably shorter, 0.75 μ to 3.48 μ in total
length. The present findings of 2n = 50 chromosomes in L. rohita is in
agreement with the earlier reports (Tripathy et al.,
2010; Nagpure et al., 2001; Khuda-Bukhsh
and Tiwary, 1994). During slide preparation bringing the chromosomes in
the same focal plane of the objective lens as well as spreading of the chromosomes
posed a major problem. Cells tend to suffer breakage easily causing unavailability
of intact chromosome complements. Because the small size and higher number of
chromosome of the complement is further disadvantageous for cytological studies
in the present material. To attain such condition one or more chromosomes would
tend to break and overlap apart in the centromere region. Therefore, utmost
care had to be exercised and practiced during slide preparation and selection
of chromosome plates in undistorted condition. However, cell preparations for
such observations must be satisfactory enough so as to avoid any misconception.
Standard haploid karyotype is formulated in the present research consisted
of 18m+6sm+1st. The karyotype study of L. rohita has been described previously
by some authors. Tripathy et al. (2010) described
the karyotype composition as 8m+12sm+22st+8t/a; Nagpure
et al. (2001) depicted the karyotype composition as 10m+14sm+8st+18t
and FN = 74; Khuda-Bukhsh and Tiwary (1994) observed
as 18m+8sm+24t and FN = 76; all of these studies reported the diploid number
to be 50.
The variation of result is dependent on several causes such as the methods
of chromosome preparation, staining procedure, tissue source of the body where
the dividing cells are found. In the present study the methodology of chromosome
preparation was different from previous studies. The result varied due to the
degree of contraction and spreading of chromosomes. Probably the difference
was due to variation in the degree of contraction of the chromosomes and methodological
constraints arising from the small size of chromosomes. Fish karyotypes are
not identical, as in human being and other plant and animal species, so a researcher
cannot have a standard karyotype for fish because not only there are differences
between species, polymorphism often occurs within the same fish species (Al-Sabti,
1991). The problem encountered is the morphological variation that exists
even between homologous chromosomes in the same nucleus (Levan
et al., 1964; Al-Sabti, 1991).
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.
1: Ahmad, Q.N., E.J. Britten and D.E. Byth, 1983. A quantitative method of karyotypic analysis applied to the soybean, Glycine max. Cytologia, 48: 879-892.
2: Alam, M.S., M. Jahan, M.M. Hossain and M.S. Islam, 2009. Population genetic structure of three major river populations of Rohu, Labeo rohita (Cyprinidae: Cypriniformes) using microsatellite DNA markers. Genes Genom., 31: 43-51.
3: Al-Sabti, K., 1991. Handbook of Genotoxic Effects and Fish Chromosome. J. Stefan Institute, Ljubljana, Yugoslavia, ISBN-13: 9788680023175, Pages: 221.
4: DoF, 2011. Jatiyo matshapakha sankalon 20-26th July. Department of Fisheries, Ministry of Fisheries and Livestock, Government of People's Republic of Bangladesh, Matshya Bhabon, Dhaka, pp: 136.
5: Kirpichnikov, V.S., 1981. Genetic Bases of Fish Selection. Springer Verlag, New York, Pages: 342.
6: Khan, T.A., M.P. Bhise and W.S. Lakra, 2000. Chromosome manipulation in fish-a review. Indian J. Anim. Sci., 70: 213-221.
Direct Link |
7: Khan, M., M. Rahman, M.S. Alam and M.M.H. Bhuiyan, 2006. Allozyme variation of hatchery and river populations of rohu (Labeo rohita, Hamilton) in Bangladesh. Aquacult. Res., 37: 233-240.
8: Khuda-Bukhsh, A.R. and S. Tiwary, 1994. Study of G-bands in two species of fish Anabas testudineus and Labeo rohita. J. Inland Fish. Soc. India, 26: 47-50.
9: Levan, A., K. Fredga and A.A. Sandberg, 1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201-220.
CrossRef | Direct Link |
10: Nagpure, N.S., B. Kushwaha, S.K. Srivastava and A.G. Ponniah, 2001. Comparative cytogenetic studies in Indian major carps Labeo rohita, Catla catla and Cirrhinus mrigala (Cyprinidae, Pisces). Chromosome Sci., 5: 153-158.
Direct Link |
11: Sarder, R.I., 2007. Freshwater Fish Seed Resources in Bangladesh. In: Assessment of Freshwater Fish Seed Resources for Sustainable Aquaculture, Bondad-Reantaso, M.G. (Ed.). FAO, Rome, Italy, pp: 105-128.
12: Sofy, H.L., A.M. Layla and M.K.A. Iman, 2008. Karyotypic diversity of some tilapia species. Nat. Sci., 6: 19-27.
Direct Link |
13: Talwar, P.K. and A.G. Jhingran, 1991. Inland Fishes. Oxford and IBH Publishing Co., New Delhi, India, Pages: 66.
14: Tan, X., J. Qin, B.N. Chen, L. Chen and X. Li, 2004. Karyological analyses on redclaw crayfish Cherax quadricarinatus (Decapoda: Parastacidae). Aquaculture, 234: 65-76.
Direct Link |
15: Thorgaard, G.H. and J.E. Disney, 1990. Chromosome Preparation and Analysis. In: Methods for Fish Biology, Schreck, C.B. and P.B. Moyle (Eds.). Chapter, 6. American Fisheries Society, Bethesda, Maryland, pp: 171-190.
16: Tripathy, S.K., K.K. Gaur and N. Sarangi, 2010. Cytogenetic characterization of B1R and C1F2 backcross progenies of Catla catla (Ham.) and Labeo rohita (Ham.). J. Occup. Health, 10: 34-37.