A method for designing primer sets for speciation studies in filamentous ascomycetes

A simple method is described for designing primer sets that can amplify specific protein-encoding sequences in a wide variety of filamentous ascomycetes. Using this technique, we successfully designed primers that amplified the intergenic spacer region of the nuclear ribosomal DNA repeat, portions of the translation elongation factor 1 alpha, calmodulin, and chitin synthase 1 genes, and two other genes encoding actin and ras protein. All amplicons were sequenced and determined to amplify the target gene. Regions were successfully amplified in Sclerotinia sclerotiorum and other sclerotiniaceous species, Neurospora crassa, Trichophyton rubrum, Aspergillus nidulans, Podospora anserina, Fusarium solani, and Ophiostoma novo-ulmi. These regions are a potentially rich source of characters for population and speciation studies in filamentous ascomycetes. Each primer set amplified a DNA product of predicted size from N. crassa.