Structural comparative analysis of T2T genome assemblies of Colletotrichum lini with different sets of chromosomes

Background: The phytopathogen Colletotrichum lini is the causative agent of flax anthracnose. Its genome has accessory chromosomes, which, as shown in other species of the genus, can determine pathogenicity. However, the structural organization, degree of variability, and evolutionary relationships of these chromosomes in C. lini have remained virtually unexplored, since their analysis requires the presence of whole-genome assemblies of the telomere-to-telomere (T2T) level. The present study aims to fill the knowledge gap regarding the genomic diversity of C. lini by comparative analysis of T2T assemblies.

Aim: To study the structural diversity of the C. lini genome and to investigate the differences and relationships between the accessory chromosomes of C. lini strains with different sets of chromosomes.

Methods: The assembly of T2T-level genomes was carried out using sequencing data we had previously obtained via the Oxford Nanopore Technologies and Illumina platforms, utilizing Canu or Hifiasm assemblers followed by Racon, Medaka, and POLCA (after Canu) or Pilon (after Hifiasm) polishing tools. The quality of the assemblies was assessed using BUSCO, QUAST, and Merqury. Comparative genomic analysis was performed by whole-genome BLAST alignment with visualization of synteny in the syntenyPlotteR R package. Phylogenetic relationships were reconstructed based on complete genome sequences by the SANS program with the inclusion of genomes of other species of the genus Colletotrichum with the "complete" assembly level available in the NCBI Genome database.

Results: The genomes of eight C. lini strains with a length of 53.7-54.7 Mb were assembled to the chromosome level: 12-14 chromosomes, BUSCO ≥98.7%. The core chromosomes (1-10) were highly conserved (size variability ≤3%), while the accessory ones (11-14) were rather variable (variability up to 38%). In strains without chromosomes 13 and 14, an increase in the size of chromosomes 11 and 12 was found. Major rearrangements were detected, including an inversion in chromosome 6 of strain #771 and inversions in accessory chromosomes of strain #391-4. Phylogenetic analysis of complete genomes showed clustering of C. lini strains in accordance with the set of accessory chromosomes. The closest species to C. lini was confirmed to be C. destructivum.

Conclusion: The study revealed a two-component architecture of the C. lini genome and demonstrated that intraspecific diversity is determined primarily by the set of accessory chromosomes, while the core chromosomes are structurally stable.