GENETIC CHARACTERIZATION OF
SELECTED FRESHWATER FISH SPECIES
ENDEMIC TO INDIAN REGION OF THE INDOBURMA BIODIVERSITY HOT-SPOT USING
ADVANCED CYTOGENETIC MARKERS
Gusheinzed Waikhom1*, Labee Thoidingjam1, Vishwamitra Singh Baisvar2,
Sukham Sanjabihari Singh1, Ravindra Kumar2 and Basdeo Kushwaha2
Institute of Bioresources and Sustainable Development, Imphal
Manipur, 795001 India
2 National Bureau of Fish Genetic Resources, Lucknow, 226002 India
1
OBJECTIVES:
 Cytogenetic investigation in fishes of IndoBurma region using conventional tools
 Development of fluorescence in situ
hybridization (FISH) tool for physical mapping
of DNA regions on fish chromosomes.
Aquaculture 2016, Las Vegas, February 22-26, 2016
NE India
Aquaculture 2016, Las Vegas, February 22-26, 2016
Rich diversity of ichthyofauna
 ~300 species
 119 genera
 38 families
Fish chromosomes characterized by large number of
small chromosomes
Only 69 species (23%) cytogeneticaly characterized
Need for bridging gap between morphological &
karyological information
Aquaculture 2016, Las Vegas, February 22-26, 2016
COLLECTION SITES
Aquaculture 2016, Las Vegas, February 22-26, 2016
Species collected
Barilius bendelisis
Barilius ngawa
Esomus danricus
Devario aequipinnatus
Devario yuensis
Amblypharyngodon mola
Osteobrama belangeri
Osteobrama cunma
Puntius chola
Tor tor
Pethia meingangbii
Schistura chindwinica
Species collected…
Schistura manipurensis
Schizothorax sp.
Neolissochilus stracheyi
Mystus ngasep
Gagata dolichonema
Glyptothorax ventrolineatus
Badis badis
Schistura chindwinica
Anabas testudineus
Trichogaster labiosus
Channa gachua
Mastacembelus armatus
 Species identification - Vishwanath et al., (2007)
 Chromosome preparations from kidney as described
by Manna and Prasad
 Conventional hypotonic spreading method & stained
with 6% Giemsa.
 Slides mounted with DPX - bright field microscope
and photographed for karyotypic analysis.
 Karyotyping followed the rules of Levan et al., 1964.
Aquaculture 2016, Las Vegas, February 22-26, 2016
Cytogenetic investigation in fishes of Indo-Burma region using
conventional tools
Devario aequipinnatus (McClelland, 1839)
Devario yuensis (Arunkumar & Tombi, 1998)
Mystus ngasep Darshan, Vishwanath, Mahanta & Barat, 2011
Pethia meingangbii Arunkumar & Tombi Singh, 2003
Pethia meingangbii
Physical mapping of genes (5S & 18SrDNA)
Barilius ngawa (Vishwanath & Manojkumar, 2002)
a
c
b
d
e
(a) Images of Barilius ngawa, (b) metaphase spread, (c) karyotype,
(d) Chromomycin A3 positive site, and (e) 5S rDNA signal.
Aquaculture 2016, Las Vegas, February 22-26, 2016
Barilius bendelisis (Hamilton, 1807)
(a) Images of Barilius bendelisis, (b) metaphase spread, (c) karyotype,
(d) AgNOR stained (one pair NOR) metaphase spread, and (e) Chromomycin A3
stained (one pair NOR) metaphase spread.
Aquaculture 2016, Las Vegas, February 22-26, 2016
Neolissochilus stracheyi (Day, 1871)
a
d
b
c
e
(a) Images of Neolissochilus stracheyi, (b) metaphase spread, (c) karyotype,
(d) AgNOR stained (one pair NOR) metaphase spread, and (e) Rhodamine
labeled 18S rDNA FISH signal (one pair) on metaphase spread.
Aquaculture 2016, Las Vegas, February 22-26, 2016
Garra spp (Hamilton, 1822)
a
d
c
b
e
(a) Images of Garra spp, (b) metaphase spread, (c) karyotype,
(d) Chromomycin A3 stained (three pairs) metaphase spread, and (e) rhodamine
labeled 18S rDNA signal on metaphase spread.
Aquaculture 2016, Las Vegas, February 22-26, 2016
CONCLUSION
• Basic chromosome information needed for genetic
investigations such as hybridization & chromosomal
manipulations in fish
• Provides a complementary data source (beside the
morphological methods) for cytotaxonomy
• Evaluate status of all species to formulate
conservation strategies for the threatened species
Acknowledgment
DBT, Government of India
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Chromosome preparations from kidney (Manna and Prasad)
Specimen injected intramuscularly with 0.05% colchicine @1 ml per 100 g to arrest the
mitotic division at the metaphase stage
After 2 hours specimens sacrificed by an overdose of ethylene glycol.
The kidneys removed and placed in a hypotonic solution of 0.56% KCl.
Each kidney was homogenized with a glass tissue homogenizer and treated in
hypotonic solution for 45 min followed by fixation using fresh chilled fixative of
methanol-acetic acid mixture (3:1 V:V).
After thorough fixation, the cellular suspension centrifuged at 1,500 rpm for 10 min.
The supernatant was discarded and the cellular pellet was suspended again in the fresh
fixative and washed 3 times or until a clear transparent cell suspension was obtained.
One droplet of the cellular suspension was dropped on grease free, pre-cleaned glass
slide from a height of 60-70 cm using pasture pipette.
Immediately, the slide was swiftly passed over a flame 2-3 times and allowed to air-dry.
The slides were then kept for aging in dust free place for 2-3 days before staining with
6% Giemsa solution (Sigma) in phosphate buffer of pH 6.8 for 15 minutes, wash with
double distilled water and air dried.
Aquaculture 2016, Las Vegas, February 2226, 2016
slides were observed under Leica DM3000 microscope and screened for good
metaphase plates.
From a total of 100 mitotic spreads (50 per sex; atleast 10 per individual) exhibiting
the complete chromosome number and characteristic morphology were scanned to
determine the modal chromosome number.
Homologous pairs of chromosomes were arranged in order of decreasing size within
each morphological group and finally, karyotype was constructed on the basis of
centromere position of ten best metaphases.
Mean length of the short arm (p) and the long arm (q), and arm ratio (the ratio of the
long arm to the short arm length) of each chromosome were calculated to classify the
chromosomes as metacentric (m), submetacentric (sm), subtelocentric (st) and
acrocentric (a), following Levan et al.,.
Fundamental number of chromosome arms (NF) was established by assigning a value
of one to all acrocentric chromosomes and a value of two to all metacentric and
submetacentric chromosomes.
Aquaculture 2016, Las Vegas, February 2226, 2016
Amplified 5S (200bp long) & 18S (1849bp long) rDNA was purified using Gel extraction
kit (Nucleopore) using recommended protocol. One microgram of purified
18SrDNAamplicons was labeled with flourescien-12-dUTP and rhodamine-5-dUTP using
nick translation protocol, according to manufacturer instructions.
Pellet of labeled 5S and 18S rDNAs were dissolved in 20-µl autoclaved ultrapure water
and check in 1% agarose gel (18S rDNA) and 2% agarose gel (5S rDNA) with 50-bp
ladder (Fermentas) for 45 minute at 80 V in Genie electrophoresis unit.
On agarose gel, the smear intensity of the probe ranged from 50-bp to 500-bp with
higher intensity at 150 to 200-bpfor 18S rDNA and 200-bp for 5S rDNA.
Finally, the probe was stored at -20°C till further use.
Aquaculture 2016, Las Vegas, February 2226, 2016
Fluorescence in situ hybridization (FISH) was performed to determine the localization of 5S
and 18S rDNAs on the metaphase chromosome. About 5 to 15 days aged metaphase
chromosomes were baked in thermal block at 90°C for 1-h as per the FISH protocol
(Winterfeld and Roser, 2007), its treat with 0.005% pepsin and denatured with 70%
formamide with 2XSSC and 0.5M EDTA, Chromosomes dehydrated with series (70%, 90% and
100%) of ethanol for each 1-2 minute at room temperature.
Hybridization of rDNA probes, mixed with formamide, dextran sulphate, salmon sperm DNA,
was performed in moist chamber for overnight at 37°C.
After hybridization of probe on chromosomes, post hybridization washes were given.
Finally, the probes hybridized with metaphase chromosome were then counter stained with
4, 6-diamidino-2-phenylindole (DAPI) and mounted with vectrashield mounting medium
(Vector Labs), kept the slides in dark for 1-h, and then the slides were examined under
fluorescence microscope (Leica) with two band filter for simultaneous visualization of the
colors, i.e. DAPI filter for chromosomes visualization in blue color, flourescein filter for
labeled probes visualization in green color and rhodamine filter for visualization in red color.
The images captured with various filters were then superimposed on each other to get the
actual location of the rDNA on the metaphase chromosomes.
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