A ROBUST DNA AMPLIFICATION FINGERPRINTING SYSTEM APPLIED TO ANALYSIS OF GENETIC-VARIATION WITHIN FUSARIUM-OXYSPORUM FSP CUBENSE

Genetic variation among isolates of F. oxysporum f.sp. cubense (Foc) w as analysed using a DNA amplification fingerprinting (DAF) system modi fied to improve reproducibility and transportability. This analysis wa s done after determining the widest tolerance range (or 'window of rep roducibility') for each component in amplification reaction. Reproduci ble polymerase chain reactions (PCRs) were achieved with between 25 an d 250 ng of template DNA, 9-15 mu M primer, 4-6 mM MgCl2 and 2-4 units of Stoffel Fragment enzyme. For experimental work we used the middle value of these ranges which allowed at least 20% error tolerance for e ach component. Similarly, thermocycling and electrophoresis conditions were also improved. Manual scoring of the DNA fingerprints was compar ed to analysis of scanned gel images using the Gel Compar program (App lied Maths, Kortrijk, Belgium). The data were clustered by unweighted pair group method analysis (UPGMA) based on the Jaccard similarity coe fficient. Isolates of Foe representing all known vegetative compatibil ity groups (VCGs) were examined and the genetic relationships between the VCGs were determined. Isolates of Foc were divided into two major groups with 30% genetic similarity. These optimized DNA amplification, thermocycling, and electrophoresis conditions were suitable for analy sis of other organisms and should be applicable to other techniques th at use arbitrary primers such as random amplified polymorphic DNA (RAP D) and arbitrarily primed-PCR (AP-PCR).