Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes

Abstract

We have compared the use of DNA sequences from the genomic internal transcribed spacer (ITS) ribosomal RNA region, with a newer method, the amplified fragment length polymorphism (AFLP) technique. ITS sequences encompass only a small part of the genome but normally reveal sufficient variability to distinguish isolates at the genus and often the species level. Although the AFLP technology reveals genome-wide restriction fragment length polymorphisms, it has not been employed extensively in establishing phylogenetic relationships. We have adapted the AFLP technology for fungal genomes and compared AFLP fingerprints generated from several fungal species and isolates from the order Ustilaginales: Ustilago hordei, U. nigra, U. aegilopsidis, U. avenae, U. kolleri, U. bullata, U. nuda, U. tritici, U. maydis, U. scitaminea, Sporisorium reilianum, and Tilletiales: Tilletia indica and T. walkeri. Geographical isolates of U. hordei and related species, particularly those infecting small-grain cereals, were difficult to distinguish when comparing ITS sequences, but were clearly separated when comparing AFLP fingerprints. The abundance of polymorphisms makes the AFLP technique more suitable to distinguish organisms in clusters of closely related species and at the isolate level. Phylogenetic analyses of the data sets generated with the two methods revealed that the AFLP-derived phylogenetic relationships were not in disagreement with the ITS-derived tree. The fungal phylogenetic tree correlated additionally with one from the graminaceous hosts generated from literature data, suggesting coevolution of some specialized host-pathogen systems. The clustering of small grain-infecting smuts due to limited genetic variability, in combination with other molecular, mating and literature data, suggests reclassification of this group possibly to include varietas designations to define host range.