Bt Research, 2015, Vol.6, No.8, 1-15
7
isolates streaked on LB agar medium (Bravo et al.,
1998). After 12 hours of incubation at 30°C, a single
colony, transferred into 100
µ
l of sterile de-ionized
water in a microfuge tube, was vortexed and kept at
-70°C for 30 min. It was then incubated in boiling
water for 10 min to lyse the cells and brie fly
centrifuged for 20 s at 12,000×g (Eppendorf
centrifuge, 5415D). The upper aqueous phase
transferred into sterile microfuge tubes was used as
template and preserved at -30°C for further use.
50-100 ng of DNA from this suspension was used as
template in RAPD- PCR analysis.
2.3 RAPD-PCR analysis
RAPD-PCR was performed using the primer (OPA 03:
5'- AGCTCAGCCA- 3') with minimum 60% G+C
content and devoid of any internal repeat as maximum
polymorphism was reported for this decamer (Kumar
et al., 2010). PCR was carried out within a reaction
volume of 25 µl [1× PCR Master mix (Promega,
USA), 2.0 μM of primer, 50-100 ng of template DNA]
in a thermal cycler (MJ mini, BioRad, USA) by 35
cycles (95°C for 1 min, 40°C for 1 min, 72°C for 1
min) with an initial denaturation step at 95°C for 4
min and a final extension step at 72°C for 15 min
(Kumar et al., 2010). PCR products (15 μl) were then
analyzed in 1.5% (w/v) Agarose (Promega, USA) gel
by horizontal electrophoresis at 60V for 1h in 1×TBE
[89 mM Tris (pH 7.6), 89 mM boric acid, 2 mM
EDTA] buffer and gel images were captured after
visualization against UV trans-illumination in a gel
documentation system (Alpha imager mini, USA)
following staining in Ethidium Bromide (EtBr)
(Sigma, USA) solution (0.5 μg/ ml) and destaining in
distilled water. Molecular weight of the DNA bands in
those gels was then determined by using Alphaview
SA software (version 3.4.0.0).
2.4 Data analysis and dendrogram construction
Binary matrix was prepared for each strain from the
gel images based on the presence or absence as scored
1 or 0 respectively for the amplicons bands. Based on
the binary matrices, similarity and distance matrices
were calculated following dice coefficient method.
These data were used in cluster analysis by
UPGMA method to construct the dendrograms
(
/). For maximum
accuracy of comparison, all isolates were processed
with the same batch of PCR master mix.
2.5 Genotyping and estimation of diversity index
Throughout the whole study, threshold level was
chosen at 0.2 in the scale bar. Each cluster was
considered a separate genotype if distances among the
strains in that cluster were less than 0.2 in scale bar.
Thus the genotypes were identified among the tested
strains as a whole and also in terms of biotype and
location. Again, the ratio between the number of
clusters and isolates for a set of strains was considered
as their diversity index. Based on this criterion, the
diversity index for biotypes and locations were
estimated and compared.
2.6 Detection of subgroups of
cry
1 genes
DNA templates (50-100 ng) from the
Bt
strains (n=
177) were mixed with PCR reaction mixture
containing 0.5 µM of each primer, 1× PCR Master
mix (Promega, USA) in 25 μl reaction volume and
amplification was performed in a DNA thermal cycler
to detect
cry
genes belonging to the
cry1
family
(major group) such as
cry1Aa
,
cry1Ac
,
cry1Ba
,
cry1Ca
. For all primer sets, PCR was carried out with
an initial single denaturation step at 95°C for 2 min
and 30 amplification cycles including denaturation at
95°C for 45 s, annealing at 53°C for 45 s and extension at
72°C for 60 s. Finally an extra extension step was
applied at 72°C for 10 min. PCR products (10 μl)
were then electrophoresed in 1.5% agarose (Promega,
USA) gel prepared and submerged in 1×TBE buffer at
60V for 60 min. Gel was visualized in a gel documentation
system following staining and de-staining.
2.7 Determination of distribution of
cry
genes in
the genotypes
Detection of
cry
genes from major groups such as
cry1
,
cry2
,
cry3
,
cry4
,
cry8
,
cry9
and
cry10
was
reported previously (Shishir et al., 2014). Certain
other subgroups of
cry
genes belonging to the
cry1
family (major group) such as
cry1Aa
,
cry1Ac
,
cry1Ba
,
cry1Ca
were also investigated. Combining the
presence of the above mentioned genes,
cry
gene
profile was obtained for each strains. Again, each
strain for its RAPD profile belongs to a cert ain
genotype. Thus, the number of
cry
genes in each
genotype and their distribution was determined.
