Application of RAPD technique to study polymorphism among Bacillus thuringiensis isolates from Jordan (original) (raw)
Nepal Journal of Science and Technology, 1970
Random Amplified Polymorphic DNA (RAPD) is a method of producing a genetic fingerprint of a particular species without its prior genetic information. Relationship between species may be determined by comparing their unique fingerprint information. B. thuringiensis was isolated from soil samples of Khumbu base camp of Everest region, Nepal. Crystal protein (delta endotoxin) producing strains (46 from Phereche and 40 from Sagarmatha national park) were tested against a series of 100 decamer RAPD primers (codes 201-300, obtained from University of British Columbia) by RAPD PCR. Primer 284 was found the best among the tested primers and the reaction condition for PCR was optimized with a PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl2 with pH 8.3.; 200ìm dNTPs each, 1U Taq polymerase , 40 pmol decamer primers, 20 ng template DNA and 1% DMSO as a final concentrations in 25ìl reaction mixture. The thermal programme was programmed as initial denaturation temperature at 94°C for ...
Bacillus thuringiensis is a rod-shaped, gram-positive, endospore-forming bacterium. It is distinguished from three other closely related Bacillus spp., viz. B. cereus, B. anthracis and B. mycoides, because of its ability to synthesize delta endotoxins (Cry proteins) as protein inclusion crystals during sporulation . The presence of a parasporal crystal, which is outside the exosporium of the endospore, is indicative of production of the toxin, and serves as a marker for this species. The bacterium was initially discovered as a pathogen of various insects and was fi rst used as an insecticidal agent. It is found in soil where it leads a saprophytic existence, but becomes an opportunistic pathogen of insects when ingested. The delta-endotoxin causes midgut paralysis and disruption when ingested by the insect host. The specifi c activities of the toxin towards insects and nontoxic nature toward animals have made this organism a useful biocontrol agent. The insecticidal crystal (Cry) J. Gen. Appl. Microbiol., 58, 83 94 (2012) Bacillus thuringiensis is a bacterium of great agronomic and scientifi c interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specifi city. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specifi c origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.
Optimization of RAPD-PCR for discrimination of different strains of Bacillus thuringiensis
Romanian …, 2009
A randomly amplified polymorphic DNA fingerprinting assay has been optimized that discriminate different B. thuringiensis isolates and serotypes. Due to advance in molecular biology technique, large numbers of highly informative DNA markers have been developed for the identification of genetic polymorphism. In the last decade, RAPD technique has been one of the most commonly used PCR based molecular markers. Relationships between species may be determined by comparing their unique fingerprint information, which are expected to be identical among related species. The optimization of certain parameters like PCR buffer, magnesium concentration and suitable primer, which produces discriminatory and reproducible fingerprints of B. thuringiensis isolates, was achieved. In addition an alkaline lysate method was optimized which give reproducible results.
Characterization of native Bacillus thuringiensis strains by PCR-RAPD based fingerprinting
Indian Journal of Microbiology, 2010
Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.
Genetic Diversity of Indigenous Bacillus thuringiensis Strains by RAPD-PCR to Combat Pest Resistance
Genetic diversity is highly relevant and significant in discovering novel insecticidal genes in Bacillus thuringiensis (Bt) strains and to deal with the problems of emerging insect resistance towards Bt biopesticides. In view of this, Random Amplified Polymorphic DNA (RAPD)-PCR analysis was performed with a decamer AGCTCAGCCA for molecular typing of 177 Bt strains of Bangladesh to determine their genetic diversity. These Bt strains were allocated into 15 genomic types with their binary matrices as determined from the dendrogram based on a standardized distance in scale bar. Genotype 9 and 11 were the largest among others, each containing more than 25% of the Bt strains. The average diversity index, as deduced for each group by cluster: isolate ratio at a specific distance, was higher for locations (0.27 ± 0.098) than that for biotypes (0.23 ± 0.046) which indicates an unmingled and vertical transfer of biochemical properties among the strains. Prevalence of agriculturally important subgroups of cry1 gene in indigenous Bt strains was also determined where cry1Aa and cry1Ca gene were found to be the most prevalent (21.74%). While analyzing the distribution pattern of cry genes, they were observed to be present in all RAPD- genotypes but genotype 10 and were most prevalent in genotypes 1, 6 and 9. The phylogeny reconstruction among the strains was performed by neighbor-joining method with the 16S rRNA gene sequences and the correlation among the phylogeny, RAPD genotypes, Biotypes and presence of cry genes were analyzed.
Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD
RAPD based fingerprinting of 2 I serovars of Bacillus tlwringiensis (Bt) representing different serotypes was performed usin g 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 16 I-2789 bp, were amplified from 13 of the I9 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two maj or clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between se-rovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.
Different techniques were adopted for molecular characterization of several indigenous strains of Bacillus thuringiensis (Bt) previously isolated from Egyptian soil samples. These isolates show different toxicity levels against neonate larvae of both insect species; Spodoptera littoralis (Biosduval); and Helicoverpa armigera (Hu¨bner). The parasporal crystals among the most potent isolates contained polypeptides of about 127 and 130 kDa. PCR screening for genes encoding different Cry genes was performed. The Cry 1 gene is the most abundant in these isolates (83.33%) among tested Cry-type genes, followed by Cry 1 gene subfamilies (Cry 1B and Cry 1C) with percentage of 38.88% and 77.77%, respectively. The tested isolates showed the presence of Cry 2A(a,b) gene, but not all of these isolates were positive for Cry 2 gene (55.55%). Only 27.77% and 16.66% of the tested isolates harbor Cry 4 and Cry 3 genes, respectively. All strains were negative in PCR assays for the Vip 3Aa1 gene. Moreover, DNA fingerprinting using RAPD-PCR was performed to detect the genetic similarities and dissimilarities among the different isolates and standard strains. Assessment of Bt diversity based on the combined analysis of their protein and RAPD-PCR banding patterns was performed. This study demonstrates that Bt strains isolated from Egyptian soil samples can be distinguished and identified on the basis of the distribution of Cry-type genes and RAPD fingerprints.
Molecular characterization of local Bacillus thuringiensis strains recovered from Northern Jordan
Journal of Basic Microbiology, 2002
A total of 80 isolates of Bacillus thuringiensis were recovered from different habitats of Northern Jordan. These isolates were grouped into three classes based on crystal morphology (spherical, bipyramidal, and both bipyramidal and cuboidal). The isolates that produced spherical crystals were the most common and the most toxic to diptera. SDS-PAGE analysis of the isolates and some reference strains with similar crystal morphology showed similar protein profiles with heterogeneous multiple protein components. The plasmid DNA content of the isolates in comparison with B. thuringiensis serovar israelensis gave a similar single intense DNA band.
Differentiation among strains and serotypes of Bacillus thuringiensis by M13 DNA fingerprinting
Journal of General Microbiology, 1991
The inter-and intraserotypic variations of Bacillus thuringiensis were studied by M13 DNA fingerprinting. Strainspecific patterns were obtained. The degree of homology was evaluated on the basis of pairwise comparisons and calculation of similarity indexes. Some strains belonging to the same serotype showed highly similar patterns, but others differed significantly. A high degree of polymorphism was established among the serotypes. These results provide evidence that the classification of B. thuringiensis strains on the basis of flagellar antigens does not always adequately reflect their genetic relatedness. DNA fingerprinting could help in future numerical taxonomic analysis of this species.