Bt Research, 2015, Vol.6, No.8, 1-15
2
Infante et al., 2010) and hemolysins (Gominet et al.,
2001; Nisnevitch et al., 2010) etc that influence the
toxicity, are also variable among the strains due to the
genetic variations beside the insecticidal proteins.
Efforts should, therefore, be continued in discovering
more
Bt
strains with genetic diversities to discover
novel toxins with improved activity, to widen the
spectrum and to overcome the resistance problems
besides protein engineering with the existing pool. So,
a good number of
Bt
strains were previously isolated
by us from different regions of Bangladesh and
studied in terms of their abundances, distribution
patterns as well as diversities connected t o
biochemical properties, plasmid and
cry
genes profile
(Shishir et al., 2014). Potentiality of these strains,
presumed through insecticidal gene and protein profile
analysis, was established for certain strains against
Melon fruit fly (
Bactrocera
cucurbitae
) upon bioassay
and this novel toxicity was reported (Shishir et al.,
2015).
Thus, more
Bt
strains and the genetic diversity
analysis among them is highly significant for
maximum utilization of the resources and to combat
the resistance problem. Several different techniques
were reported for genetic diversity analysis, like M13
fingerprinting (Miteva et al., 1991), arbitrary primer
PCR (Brousseau et al., 1993), PCR using conserved
primers for 16S to 23S ribosomal intergenic spacer
sequences (Bourque et al., 1995), DNA hybridization
using variable region of 16S rRNA gene (te Giffel et
al., 1997), AFLP fingerprinting, RAPD-PCR (Welsh
and McClelland, 1990) etc. Ribotyping, either by PCR
or DNA hybridization, failed to detect the diversity
among
Bt
strains which could be because of the use of
one single gene or operon and the evolutionarily
conserved nature of rRNA gene. However, when the
whole genome was used for identification of
Bt
strains
by M13 DNA fingerprinting and arbitrarily primed
PCR, considerable diversity among the
Bt
serovars
representing different serotypes had been detected.
Random amplification of polymorphic DNA (RAPD)
is a modified method of PCR with a single arbitrary
primer that recognizes differences in the prevalence
and positions of annealing sites in the genome and
produces a varied spectrum of amplicons reflecting
the genomic composition of the strains (We lsh
and McClelland, 1990; Williams et al., 1990). Besides
this taxon-specific amplicon generation for the
detection of diversity, RAPD-PCR fingerprinting
technique is faster, less labor-intensive and more
reliable in comparison to other molecular typing
methods (Bostock et al., 1993; Sikora et al., 1997)
and thus was found to be more feasible method for
this study.
Considering the high relevance of the above facts, the
present study was designed to determine the genetic
diversity among 177
Bt
strains of Bangladesh and to
reveal the correlation between the distribution pattern
of
cry
genes to the diversity.
1 Results and Discussion
1.1 RAPD profile based genotyping
The specific typing of
B.
thuringiensis
enables
tracking of strains dispersed in the environment and
assist in the discovery of new strains. The existing
serotyping scheme, while having provided an
invaluable basis for
Bt
classification for a long time,
provides no information about the genetic relatedness
of strains within groups and between groups and does
not necessarily indicates the degree and spectrum of
toxicity (Gaviria- Rivera and Priest, 2003). Contrarily,
RAPD is a modified PCR method with a single
arbitrary primer that recognizes differences in the
prevalence and positions of annealing sites in the
genome producing a spectrum of amplicons that are
considered to reflect the genomic composition of the
strain and may vary along the strains (Welsh and
McClelland, 1990; Williams et al., 1990). The
advantage of this method is that no prior knowledge of
the genome under research is necessary (Bostock et al.,
1993; Sikora et al., 1997) and it was found from
several studies that the RAPD analysis could
effectively distinguish between the
Bt
strains (Kumar
et al., 2010). Hence, the analysis of genetic diversity
among the indigenous
Bt
strains was done by
RAPD-PCR method in this study.
A total of 177
Bt
strains were employed for
RAPD-PCR amplification with decamer OPA 03 (5'-
AGCTCAGCCA -3') which was reported to produce
100% polymorphism (Kumar et al., 2010) and also
observed to be efficient in few initial screenings in
this study (Data not shown). Molecular weights
of these bands were then estimated comparing with
