New smut-specific primers for multilocus genotyping and phylogenetics of Ustilaginaceae

The Ustilaginomycotina, often collectively referred to as smut fungi, represent one of the three subphyla of the Basidiomycota. Smut fungi predominantly parasitize Angiosperms, are globally distributed, and contain several economically important pathogens. The most species-rich family of the smut fungi is the Ustilaginaceae. To investigate the molecular phylogeny of smut fungi, most studies rely on nrDNA loci, such as ITS and LSU. Protein coding genes, like rpb1, rpb2, TeF1a, atp6, and ß-tubulin, have been used in some studies. However, because of the huge diversity of smut fungi and the lack of dedicated primers, amplification of these loci has proven difficult for several groups. Thus, it was the aim of the current study to develop primers for new loci for the smut fungi with the focus on the largest family, the Ustilaginaceae. Here, the development and testing of new primers for nine loci based on protein-coding genes is reported (myosin, map, rpl3, tif2, ssc1, ß-tubulin, sdh1, rpl4A and atp2). A list of various primer combinations for the amplification of the new loci is given, with the corresponding PCR conditions and the best combinations for several genera of the Ustilaginaceae and some other Ustilaginomycetes. We hope that the primers presented in this study will be useful in overcoming the limitations of currently-used loci in terms of phylogenetic resolution, especially with respect to resolving species complexes and providing a better resolution of the higher-level phylogenetic relationships of smut fungi.