Reliability of RAPD fingerprinting of three basidiomycete fungi, Laccaria, Hydnangium and Rhizoctonia

Abstract

Randomly amplified polymorphic DNA (RAPD) profiles are currently being developed for Laccaria and Hydnangium species and Rhiwcfonia solani. The technique is increasingly being used to differentiate fungal isolates. As for the polymerase chain reaction (PCR) from which it was derived, the conditions necessary for reproducible RAPD products have received attention. However, in contrast to the PCR reaction, the technique relies on non-specific primers and as a consequence the reaction conditions are not necessarily as specific as they are in PCR. Compared with PCR products, RAPD fingerprints were therefore more sensitive to reaction and thennocycle conditions. RAPD products produced using 10 and 17-23 mer primers were visualized on ethidium bromide stained p~l~acrylarnide electrophoresis gels. Factorial experiments showed RAPD patterns were altered by changes in various reaction mixture components including the concentration of non-DNA impurities, the number and concentration of primers and the DNA polymerase enzyme type, source and concentration. These factors, particularly the enzyme source, reacted differently with the magnesium chloride concentration. Fluorescent dye labelled primers were used with internal lane standards in a DNA sequencing system to assess accurately the molecular weights and relative amounts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on patterns visualized on ethidium bromide stained non-denaturing polyacrylamide gels. The temperature at which DNA was denatured in the first cycles altered the fingerprints. Reaction mixture temperatures of 9 4 ' or higher, compared with 91-93O, caused loss of visual yield of some products, particularly those greater than 500 base pairs, and increased the yield of others. Reproducibility of RAPD patterns, when the reaction mixture and temperature profile factors were varied, was facilitated by cross reference to fluorescently labelled bands separated with internal lane standards in a DNA sequencing system. Reproducibility of fingerprint patterns in a standard reaction mixture was achieved, with different thennocycle programmes and in different thennocyclers when the temperature profile was reproduced, suggesting that particular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used. DNA fingerprinting is a technique which has been widely adopted in order to differentiate among organisms at the species and subspecies levels (Maclean ef al., 1993). Restriction fragment length polymorphism (RFLP) analysis has been used successfully to distinguish closely related fungi (Forster et al., 1987; Levy ef al., 1991). This technique is time consuming and requires large quantities of DNA and specific probes. Recently Williams ef al. Welsh &McClelland (1990) described a technique which makes use of single species of arbitrary primers to amplify DNA polymorphisms (RAPD) using a modified polymerase chain reaction (PCR, Sa...

Spatial distribution of discrete RAPD phenotypes of a root endophytic fungus, Phialocephala fortinii, at a primary successional site on a glacier forefront

“…The samples from 1993 and 1994 were analysed separately. The loci from the two separate samplings were not considered comparable because of potential reproducibility problems (see Meunier & Grimont, 1993 ;Tommerup et al, 1995). Consequently, continuity of the identical isolates detected in 1993 was tested by side-by-side comparison with the samples from 1994.…”

Genetic Diversity Analysis of Jatropha Curcas Provenances Using Randomly Amplified Polymorphic DNA Markers

“…This lack of specificity was further exacerbated by the low stringency during PCR that promotes the amplification of the non-specific products. Several PCR variables were known to improve RAPD consistency, including template DNA purity, consistent primer concentration, a single source of DNA polymerase between reactions, and the use of a single thermocycler set at a certain reaction profile (Tommerup et al, 1995). All those factors were observed during this study, but these optimizations by no means guarantee that the DNA bands, which were visualized on gels, would be free from false positives and negatives.…”

Molecular markers in ecology of ectomycorrhizal fungi

“…A key assumption about genet identification is that DNA fragment sizes can be identified unambiguously. This can be a problem with RAPD, since some bands are difficult to amplify reproducibly (Tommerup et al, 1995). If the PCR conditions are defined stringently (annealing temperature = 55 °C), RAPD amplification is usually reproducible (Selosse et al, 1998).…”