International Journal of Aquaculture, 2015, Vol.5, No.27 1
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8
reported that the genetic diversity of this fish
population was low (Mukhopadhyay T, Bhattacharjee,
2014a). Therefore,
a limited number of individuals
were involved for further population genetic analyses
p
rimarily because of the dwindling population
structure of this species in the study region. The
collection spots were as follows: TR-1, TR-2, TR-3,
TR-4, TR-5 and TR-6. The local names and
geographical co-ordinates of the collection spots are
mentioned in Figure 1. Fishes were identified according
to Talwar and Jhingran (1991).
3.2
Isolation of high molecular weight DNA and
Quantification
Genomic DNA (gDNA) was extracted noninvasively
from tiny amount of tissue samples (10-15 mg of fin
clips from the caudal and ventral portions and/or
25-30 pieces of scales from the dorsal portion) from
the live
Badis
badis
following Mukhopadhyay and
Bhattacharjee (2014b).
Briefly, tissues were chopped
finely and then incubated at 52ºC for 60 min in a lysis
buffer containing 15 µl Proteinase K (20 mg/ml) and
30 µl 20% SDS. After the incubation, each lysate was
purified by phenol:chloroform:isoamyl alcohol
(25:24:1, v/v/v).
3.3
Primer Selection
Forty arbitrary decamer primers of random sequences
(Kit-A and Kit-B, twenty primers from each kit) were
purchased from Imperial Life Science Pvt. Ltd., India.
Firstly, six different populations were screened with
the forty primers and finally twenty-two (10 primers
from Kit-A and 12 primers from Kit-B) were selected
for further analyses on the basis of the variability and
reproducibility of the bands obtained (Table 1).
3.4
RAPD-PCR and documentation of amplified
products
RAPD analyses were performed in a 96 well Peltier
Thermal Cycler (Applied Biosystems 2720, Life
Technologies, USA) in a final reaction volume of 25
µl, each containing a final concentrations of ~100-150
ng of isolated gDNA, 1.6 pM of OPA or OPB primer,
1X Standard Taq Polymerase buffer (10mM Tris-HCl,
pH 8.3, 50 mM KCl, 1.5 mM MgCl
2
) (NEB, USA),
200 µM of each dNTPs (dATP, dTTP, dCTP, dGTP)
(NEB, USA), and one unit of Taq DNA Polymerase
(NEB, USA). PCR cycling programs were as follows:
initial denaturation at 94ºC for 5 min followed by 40
cycles of 94 ºC, 1 min for denaturation; 35 ºC, 1 min
for annealing; 72 ºC, 2 min for elongation and finally
an extension at 72 ºC for 10 min. The amplified
products were electrophoresed in an ethidium bromide
(0.5 µg/ml) pre-stained 1.4 % (w/v) agarose gel
(Lonza, Basel, Switzerland) at a constant voltage 100
V and current 100 mA in TAE buffer (40 mM
Tris-HCl, pH 8.0; 20 mM Acetic acid; 1 mM EDTA,
pH 8.0) using BenchTop Labsystems BT-MS-300,
Taiwan electrophoretic apparatus. Molecular weight
of each band was estimated using a standard 100 base
pair ladder (NEB, USA) and/or Lambda EcoRI/
HindIII double digest DNA size marker (NEB, USA).
The gels were visualized on the UV-transilluminator
(Spectroline BI-O-Vision
®
NY, USA) and photographed
using a digital camera.
3.5
RAPD data analyses
A robust RAPD dataset from six
Badis badis
populations were analyzed for assessing inter-population
genetic variability, genetic differentiation, genetic
distance and genetic hierarchy between six populations
of
Badis badis
of Terai region. The RAPD
fingerprinting profile were computed and analyzed in
the form of binary variables (1= band present or 0=
band absent) by direct comparison of the amplified
pattern. The binary scores obtained from all the
twenty-two primers in the RAPD analyses were then
pooled for constructing a single data matrix.
The
RAPD data was analysed using three software viz.,
Popgene ver. 1.32 (Yeh et al., 1999), TFPGA ver.1.3
(Miller, 1997) and GenAlEx 6.5 (Peakall and Smouse,
2006; Peakall and Smouse, 2012). The data matrix
was used to estimate the observed number of alleles or
allelic richness [
(1/K) ∑ n
i
, where
K
= number of loci
and
n
i
= the number of alleles detected per locus],
Shannon’s Information Index (
H´
or
I= -∑ p
i
log2 p
i
,
where
H´
or
I
is diversity and
p
i
is the frequency of a
particular RAPD band) (Lewontin, 1972). To calculate
the measure of evenness (
E
) we have used the
exponential function of Shannon’s Index i.e,
e
H´
and
subsequently divided it by allelic richness (S) or
observed number of alleles (
E= e
H´
/S
, where S is the
observed number of alleles or allelic richness). The
Shannon’s Information index (
H´
), measure of
evenness (
E
) and observed number of alleles i.e.
richness (
S
) sum up to
SHE analysis
(
H´= lnE+ lnS
)
(Hayek and Buzas, 1997; Magurran, 2004). To
