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Colletotrichum lini is a pathogenic fungus that infects flax and causes significant yield losses. In this study, we assembled the genomes of four highly virulent C. lini strains using the Oxford Nanopore Technologies (ONT, R10.4.1 flow cells) and Illumina platforms. The performance of two tools developed for telomere-to-telomere (T2T) genome assembly was compared: Verkko and Hifiasm. Prior to the assembly, ONT reads were corrected using the HERRO algorithm. Verkko generated genome assemblies of high completeness but low contiguity, while Hifiasm allowed the generation of T2T assemblies. Despite significantly different genome coverage with ONT data (25–100×), four assemblies of equal contiguity were obtained: 53.6–54.7 Mb, ten core chromosomes, and two or three accessory chromosomes. A comparative analysis of different polishing tools showed that at a certain genome coverage with the corrected ONT data (≥35×), the additional polishing of the assembly did not improve its accuracy, even with the Illumina data. An analysis of the genome structures of the four C. lini strains revealed a high similarity between the core chromosomes. Thus, our approach enabled assembling T2T Colletotrichum genomes only from the ONT data obtained using R10.4.1 flow cells and may be promising for other fungal genera. These assemblies will allow the accurate identification of strain-specific differences at the chromosome level and will aid in the development of effective strategies to protect flax from anthracnose. [ABSTRACT FROM AUTHOR]

Colletotrichum lini is a fungal pathogen of flax that can cause significant yield and quality losses. In this work, we obtained the first complete annotated genome assembly of the highly virulent C. lini strain #394-2. The nuclear genome consisted of ten core and two accessory chromosomes and had a length of 53.7 Mb. The mitochondrial genome was 39.1 kb. The assembly was obtained by the Canu–Racon ×2–Medaka–Polca algorithm using Oxford Nanopore Technologies and Illumina data. As a result of the annotation with the Illumina RNA-Seq data, 12,449 genes were identified. Potential signaling proteins were tested for effector functions and 550 effector proteins were predicted using EffectorP. The visualization of the effector protein localization revealed that the presence of effector proteins was associated with repeat-rich regions. A comparison of the genomic structure of C. lini with chromosome-level and complete assemblies of the genus Colletotrichum representatives revealed that the genomes of Colletotrichum species differed by the presence of chromosomal rearrangements. The obtained assembly expands the knowledge of the genomic structure of Colletotrichum species and provides the basis for further studies of C. lini, which will help to understand the virulence mechanisms and protect flax from anthracnose. [ABSTRACT FROM AUTHOR]

Colletotrichum lini is a flax fungal pathogen. The genus comprises differently virulent strains, leading to significant yield losses. However, there were no attempts to investigate the molecular mechanisms of C. lini pathogenicity from high-quality genome assemblies until this study. In this work, we sequenced the genomes of three C. lini strains of high (#390-1), medium (#757), and low (#771) virulence. We obtained more than 100× genome coverage with Oxford Nanopore Technologies reads (N50 = 12.1, 6.1, 5.0 kb) and more than 50× genome coverage with Illumina data (150 + 150 bp). Several assembly strategies were tested. The final assemblies were obtained using the Canu–Racon ×2–Medaka–Polca scheme. The assembled genomes had a size of 54.0–55.3 Mb, 26–32 contigs, N50 values > 5 Mb, and BUSCO completeness > 96%. A comparative genomic analysis showed high similarity among mitochondrial and nuclear genomes. However, a rearrangement event and the loss of a 0.7 Mb contig were revealed. After genome annotation with Funannotate, secreting proteins were selected using SignalP, and candidate effectors were predicted among them using EffectorP. The analysis of the InterPro annotations of predicted effectors revealed unique protein categories in each strain. The assembled genomes and the conducted comparative analysis extend the knowledge of the genetic diversity of C. lini and form the basis for establishing the molecular mechanisms of its pathogenicity. [ABSTRACT FROM AUTHOR]