Abstract
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.
Similar content being viewed by others
References
Hofte H and Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Micorbiol Rev 53:242–255
Charles JF, Nielson-LeRoux C and Deecluse (1996) A Bacillus sphericus toxins: Molecular biology and mode of action. Annul Rev Entomol 41:389–410
Martin P and Travers R (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl Environ Microbiol 55: 2437–2442
Iriarte J, Bel Y, Ferrandis MD, Andrew R, Murillo J, Ferre J and Caballero P(1998) Environmental distribution and diversity of Bacillus thuringiensis in Spain. Sys Appl Microbiol 21:97–106
Caetano-Anolles G, Bassam BJ and Gresshoff PM (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotige primers. Biotechnology 9:553–557
de Bruijn (1992) Use of repetitive (repetitive extragenic palandromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Applied Env Microbiol 58: 2180–2187
Sikora S, Redzepovic S, Pejic, I and Kozumplik V (1997) Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting. J Applied Microbiol 82:527–531
Welsh J and McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucl Acids Res 18: 7213–7218
Williams JGK, Kubelik A R, Livak KJ, Rafalski JA and Tingey SV (1990) DNA polymorphisms amplified by aribitary primers are useful as genetics markers. Nucleic Acids Res 18:6531–6535
Brousseau R, Saint-Onge A, Prefontaine G, Masson L and Cabane J (1993) Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl Environ Micorbiol 59:114–119
Hansen BM, Damgaard PH, Eilenberg J and Pedersen JC (1998) Molecular and phenotypic characterization of Bacillus thuringiensis isolates from leaves and insects. J Invertebr Pathol 71:106–114
Travers RS, Martin PA and Reichelderfer CF (1987) Selective process for efficient isolation of soil Bacillus sp. Appl Environ Micorbiol 53:1263–1266
Ohba M and Aizawa K (1986) Insect toxicity of Bacillus thuringiensis isolated from soils of Japan. J Inverte Pathol 47:12–20
Welsh J and McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18: 7213–7218
Yaradoni SN, Krishnaraj PU, Ussuf KK and Kuruvinashetti MS (2003) Molecular characterization of isolates of Bacillus thuringiensis from Western Ghats. J Plant Biol 30:7–13
Gaviera-Rivera AM and Priest FG (2003) Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR. Syst Appl Micobiol 26:254–261
Malkawi HI, Al-Momani F, Meqdam MM, Saadoun I and Mohammad MJ (1999) Detection of genetic polymorphisms by RAPD-PCR among isolates of Bacillus thuringiensis. New Microbiol 22:241–247
Pattanayak D, Chakrabarty SK, Kumar PA and Naik PS (2001) Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD. Indian J Exp Biology 39(9) 897–901
Bostock A, Khattak MN, Mathews R and Burnie J (1993) Comparison of PCR fingerprinting, by random amplification of polymorphic DNA with other molecular typing methods for Candida albicans. J General Microbiol 139: 2179–2184
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kumar, D., Chaudhary, K. & Boora, K.S. Characterization of native Bacillus thuringiensis strains by PCR-RAPD based fingerprinting. Indian J Microbiol 50, 27–32 (2010). https://doi.org/10.1007/s12088-009-0011-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12088-009-0011-3