Whole-genome sequencing and de novo assembly.

F. oxysporum genomic DNA was isolated through phenol-chloroform extraction from freeze-dried mycelium that was harvested from 5-day-old NO₃ medium (0.17% yeast nitrogen base, 3% sucrose, 100 mM KNO₃) cultures as described in detail in reference 33. Library preparation of insert size 550 bp and Illumina HiSeq 2500 paired-end sequencing were performed at Keygene N.V. (Wageningen, Netherlands).

Sequencing reads were trimmed for quality and to remove adapter sequences with FastqMcf v1.04.676 (https://github.com/ExpressionAnalysis/ea-utils/blob/wiki/FastqMcf.md; quality threshold = 20). De novo assemblies were generated using CLC-workbench 8.0. Default settings were used, except “minimum contig length = 500.”

For generating a core phylogeny, homologs of 15,956 Fol4287 core genes (including introns) were searched in all genomes using BLASTN with default parameters. We selected all sequences that overlapped >70% with the query sequence and with more than 80% identity to the query. We then selected query genes for which we found only a single hit in each genome, leaving us with 440 genes. We used ClustalO (60) to construct a multiple-sequence alignment for each gene and a custom python script to concatenate these alignments. This alignment was subsequently trimmed using trimAl-strictplus. We used PhyML v20120412 (61) with 100 bootstraps to infer phylogeny and ETE v3.0.0b35 (62) to visualize the tree.