High-Density Genotype-By-Sequencing of Septoria musiva: From SNP Discovery to Association Mapping of Virulence Factors

Septoria musiva is a necrotrophic fungal pathogen that causes leaf spot and stem canker of hybrid poplar. Development of durable resistance to this pathogen requires characterization of susceptibility/resistance genes in the host population and virulence genes in the pathogen population. To achieve this goal robust phenotyping and genetic characterization of the pathogen population is essential.  We have developed a protocol for S. musiva phenotyping; however, the lack of high-density genotyping of natural populations of S. musiva prevents the genetic characterization of virulence. To overcome this bottleneck, a non-resource prohibitive method of deep coverage genotyping of fungal populations is required. Here we describe the use of a two-enzyme genotype-by-sequencing (GBS) method adapted for ION Torrent sequencing technology. Using the sequenced genome of S. musiva to validate this technique 2,373 unique loci, “sequence tags”, containing 9,992 SNPs were identified and characterized from a small (n =18) natural population. Based on the estimated size of the S. musiva genome, ~30 Mb, GBS analysis placed a SNP marker on average every 12.6 kb in the fungal genome. Blast searches of the sequence tags generated from the S. musiva genome revealed that ~95% represent predicted genes, providing ~23% coverage of the total predicted gene space. The marker density generated is adequate for association mapping in natural S. musiva populations when robust virulence phenotyping data is available.