Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markers.

摘要

Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies..