Resumen

BACKGROUND Puccinia melanocephala (brown rust) and Puccinia kuehnii (orange rust) are major biotrophic pathogens severely affecting sugarcane production worldwide. Despite their economic impact, genomic resources for these species remain limited due to challenges inherent to obligate biotrophy, contamination during field sampling, and highly repetitive, dikaryotic genomes. This study aimed to generate high-quality phased genome assemblies for Colombian isolates of both pathogens and to perform structural, functional, and repeat-content analyses to provide foundational genomic resources for comparative genomics and sugarcane breeding programs. RESULTS : PacBio HiFi sequencing combined with LongPlex library preparation and phased assembly using Hifiasm enabled the generation of high-quality haplotype-resolved assemblies for both species. GenomeScope profiles indicated complex dikaryotic architectures, while BlobToolKit analyses identified and removed environmental contaminants, resulting in curated assemblies of 322.68 Mb (P. melanocephala) and 267.89 Mb (P. kuehnii). Compleasm completeness values exceeded 86% in both genomes. Transposable elements dominated both genomes, accounting for 73.7% of P. melanocephala and 68.7% of P. kuehnii, with LTR retrotransposons (Gypsy, Copia) and TIR transposons (Mutator, CACTA) being the most abundant. BRAKER predicted 32,412 and 15,690 protein-coding genes in P. melanocephala and P. kuehnii, respectively, with BUSCO completeness above 80%. Functional annotation with EggNOGmapper identified over 9,000 unique GO terms per species, and enrichment analysis of 1,849 shared genes revealed significant overrepresentation of biological processes and cellular components related to nuclear organization, intracellular signaling, protein transport, and fungal-type cell wall organization. CONCLUSIONS : These genomic assemblies represent the first genomic resources for Colombian isolates of P. melanocephala and P. kuehnii. The strong TE-driven genome expansion, diverse gene repertoires, and enriched functional categories highlight the evolutionary dynamics underlying pathotype diversification and host adaptation. These genomic resources provide a foundation for effector discovery, comparative genomics, and molecular surveillance, and offer valuable tools for sugarcane breeding programs aimed at developing durable resistance against rust pathogens.