Genomic differences among three Hemileia vastatrix races: exclusive clusters and functional classes

Hemileia vastatrix, the causal agent of Coffee Leaf Rust (CLR), represents the principal challenge to Arabica coffee production. The dynamic co-evolution system within the H. vastatrix–Coffea pathosystem promotes the emergence of novel pathotypes with enhanced virulence, capable of overcoming host resistance mechanisms. One of the main challenges for advancing the development of cultivars with durable resistance to CLR is the limited genomic information available for H. vastatrix, given the diversity of races that have already been identified. Thus, the aim of this study was to compare the protein groups of three contrasting H. vastatrix races II, XIII, and XXIII, to identify sets of proteins unique to each genotype. To achieve this, the genomes of H. vastatrix races II and XIII were sequenced and annotated. Genomic DNA was extracted from urediniospores of each isolate and sequenced using Illumina MiniSeq System. Adapters sequences were trimmed using Trimmomatic, and the quality of the filtered data was assessed with FastQC. A draft genome assembly was generated using SPAdes v3.15.5. Scaffolds were obtained with RagTag v2.1.0. Assembly statistics and genome completeness were evaluated using QUAST and BUSCO v5.0.0, respectively. Structural gene prediction was performed using AUGUSTUS via the BRAKER2 pipeline. The predicted proteomes of races II and XIII were compared with that of race XXIII using OrthoFinder and OrthoVenn3. The draft genome assemblies exhibited high contiguity, with N50 values of 5.4 Mb for race II and 7.3 Mb for race XIII, and maximum contig sizes of 13.7 Mb and 18.3 Mb respectively. Functional enrichment analysis of proteins exclusive to three H. vastatrix races revealed similar patterns, but with significant differences. Regarding molecular functions often associated with host-pathogen interactions, race XIII displayed exclusive genes with additional functions such as oxidoreductase activity, ion binding, and transferase activity, suggesting greater functional diversity. Race XXXIII also had exclusive genes for hydrolases, transferases, peptidases, and oxidoreductases, and additionally included functions such as motor activity, helicase, and monooxygenase activity, which were less represented in the other races. Race II had fewer exclusive genes but maintained predominance in hydrolases, transporters, and peptidases. These exclusive gene sets highlight functional differences that may influence host-pathogen interactions. The results confirm that distinct races possess specific resistance strategies. Characterizing these genes can provide valuable insights for developing resistant cultivars and for monitoring pathogen populations.