Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution

Background Accessory genome regions of plant pathogenic fungi, which are highly variable and consist of niche-adaptive genes, play a crucial role in shaping host-specific interactions but are notoriously difficult to assemble. Fusarium oxysporum causes some of the world’s most economically devasting diseases, however, understanding how it interacts with its host is hindered by challenges in assembly of accessory genome regions/chromosomes, even with long read sequencing technologies. F. oxysporum f. sp. lactucae (FOLac) races 1 and 4 possess highly similar core genomes but cause distinct virulence phenotypes on specific lettuce cultivars. The availability of fully assembled genomes for the two races is needed to advance our understanding of the genetic basis of pathogenicity and the evolutionary processes underlying the diversification of FOLac and other F. oxysporum pathogens. Results We developed an assembly workflow for generating gapless, telomere-to-telomere (T2T) complete genome assemblies for FOLac races 1 and 4. The T2T assemblies allowed for the identification of 16 chromosomes (5 accessory) and 20616 predicted genes for race 1 and 19 chromosomes (8 accessory) and 20292 predicted genes for race 4. Comparative genomics revealed major structural differences in their accessory genome regions, including genome rearrangement and large-scale chromosome duplication, with results suggesting transposable elements as the main drivers of those genomic changes. The analysis of Secreted in Xylem ( SIX ) effector gene profiles uncovered a similar presence/absence pattern among FOLac races 2–4, distinguishing them from race 1. Searches for genes unique to each race resulted in the identification of 689 race 1- and 536 race 4-specific genes. Assembly and genomic features comparing T2T to contig-level Illumina assemblies showed that 17–23% of genome sizes and ~ 10% of predicted genes were missing from Illumina assembly, mostly within accessory genome regions. Conclusions T2T assemblies revealed large-scale differences in accessory genome structure and content between two otherwise highly similar pathogenic races. These differences provide a framework for understanding evolutionary processes that led to the diversification of pathogens within F. oxysporum on a fine evolutionary timescale, the identification of genes that may be responsible for host-pathogen interaction, and the identification of race-specific sequences useful for diagnostics.