DNA fingerprinting of the bacterial blight pathogen of rice using PCR-based methods
1996
George, M.L.C. | Cruz, W.T. | Nelson, R.J. (International Rice Research Inst., P.O. Box 933, 1099 Manila (Philippines))
Repetitive DNA sequences are pervasive in the genomes of most organisms. Their conserved nature and varied distribution in the genome allows the detection of many polymorphisms that are useful in differentiating individuals. IS 1112 is a high-copy number repetitive element isolated from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae. Two outwardly-directed primers complementary 20 sequences in IS 1112 were used to fingerprint a set of 71 X. oryzae pv. oryzae strains using PCR, PCR-based restriction analysis (PBRA) and ligation-mediated PCR (LMPCR). To allow amplication of long DNA fragments, standard amplification conditions were amended by increasing the pH, adding dimethyl-sulfoxide, decreasing denaturation time, and increasing extension time. Bands ranging in size from 100 bp to 7 kb, and in number from 13 to 48 bands (average of 26 in PCR, 35 in PBRA and 36 in LMPCR) were amplified. The three methods revealed useful polymorphism among individual strains and allowed their genetic relationships to be efficiently deduced. Good correlation was found between the major clusters obtained by the three methods. The bootatrap values, which indicate the strength of the groupings, are generally high for the major clusters produced, with PCR giving the most robust clusters. Overall, the PCR method was most efficient in terms of simplicity and economy. The PCR and LMPCR methods were used to fingerprint representative field isolates of the bacterial blight pathogen collected in the Philippines in 1993-1994. Similar groupings, which correspond very well to those derived using RFLP (restriction fragment length polymorphism) were obtained using PCR and LMPCR
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