Development of sequence characterized amplified genomic regions (SCAR) for fungal systematics: proof of principle using Alternaria, Ascochyta and Tilletia

Abstract

SCARs were developed by cloning RAPD-PCR amplicons into commercially available vectors, sequencing them and designing specific primers for PCR, direct sequencing and phylogenetic analysis. Eighteen to seventy percent of cloned RAPD-PCR amplicons were phylogenetically informative among closely related small-spored Alternaria spp., Ascochyta spp. and Tilletia spp., taxa that have been resistant to phylogenetic analysis with universally primed, protein-coding sequence data. Selected SCARs were sequenced for larger, population-scale samples of each taxon and demonstrated to be useful for phylogenetic inference. Variation observed in the cloned SCARs generally was higher than variation in nuclear ribosomal internal transcribed spacer (ITS) and several protein-coding sequences commonly used in lower level fungal systematics. Sequence data derived from SCARs will provide sufficient resolution to address lower level phylogenetic hypotheses in Alternaria, Ascochyta, Tilletia and possibly many other fungal groups and organisms.

• anonymous loci
• Ascochyta blight
• bunt fungi
• Didymella
• Phoma
• phylogeny
• population-level systematics
• speciation

Acknowledgments

The authors thank Drs E. Njambere, R. Attanayake, T. Drader and Ms. K.A. Thomas for excellent laboratory assistance. We acknowledge the Derek Pouchnik Laboratory for Biotechnology and Bioanalysis, Washington State University and Lisa Castlebury, USDA-ARS Systematic Mycology and Microbiology Lab in Beltsville, Maryland, for sequencing support. This work was partially supported by National Science Foundation grant DEB-0416314.