International Journal of Aquaculture, 2016, Vol.6, No.4, 1
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Table 5 The primer name, its sequences and GC content
Sl/No.
Primer
Sequence
G+ C Content
(5´
3´)
(%)
1
OPA-01
CAGGCCCTTC
70
2
OPA-02
TGCCGAGCTG
70
3
OPA-04
AATCGGGCTG
60
4
OPA-07
GAAACGGGTG
60
5
OPA-09
GGGTAACGCC
70
6
OPA-10
GTGATCGCAG
60
7
OPA-13
CAGCACCCAC
70
8
OPA-16
AGCCAGCGAA
60
9
OPA-19
CAAACGTCGG
60
10
OPA-20
GTTGCGATCC
60
11
OPB-01
GTTTCGCTCC
60
12
OPB-03
CATCCCCCTG
70
13
OPB-04
GGACTGGAGT
60
14
OPB-06
TGCTCTGCCC
70
15
OPB-07
GGTGACGCAG
70
16
OPB-11
GTAGACCCGT
60
17
OPB-12
CCTTGACGCA
60
18
OPB-15
GGAGGGTGTT
70
19
OPB-17
AGGGAACGAG
60
20
OPB-18
CCACAGCAGT
60
4.2 Genomic DNA isolation
High molecular weight genomic DNA was extracted and quantified using the protocol followed by
Mukhopadhyay and Bhattacharjee (2014).
4.3 RAPD–PCR amplification, documentation and analysis
Twenty RAPD primers from Kit-A and B (Imperial Life Science Pvt. Ltd., India) were selected for further
analyses on the basis of the numbers, variability and reproducibility of the bands obtained (Table 5). RAPD
analyses were performed in a 96 well Peltier Thermal Cycler (Applied Biosystems 2720, Life Technologies, USA)
following Mukhopadhyay and Bhattacharjee (2014). The amplified products were electrophoresed in an ethidium
bromide (0.5µg/ml) pre-stained 1.4 % agarose gel (Lonza, Basel, Switzerland) in TAE buffer using BenchTop
Labsystems BT-MS-300, Taiwan electrophoretic apparatus. The standard 100 base pair ladder (NEB, USA) were
used to approximate molecular weight of each band. The RAPD data was analysed using three software viz.,
Popgene ver. 1.32 (Yeh et al., 1999) and GenAlEx 6.5 (Peakall and Smouse, 2006; Peakall and Smouse, 2012).
The data matrix was used to estimate locus specific different diversity indices i.e.,
the observed number of
alleles (Na), effective number of alleles (Ne), percentage of polymorphic loci (P), Shannon Diversity Index (I) and
Nei’s genetic diversity (H). Inter-population genetic differentiation was estimated by pairwise PhiPT values,
where PhiPT = AP / (WP + AP) = AP / TOT (AP = Estimated variance Among Populations, WP = Estimated
variance Within Population, TOT= Estimated variance) (Excoffier et al., 1992).
Acknowledgments
The work was supported by a research grant from University Grants Commission (UGC), India [MRP Sanction No.: 40-289/2011
(SR)] awarded to corresponding author.
