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Flax (Linum usitatissimum L.) is known as a dual-purpose crop, producing both fiber and oil, which have a wide range of uses. Successful flax breeding requires knowledge on the genetic determinants of flax traits. The former identification of molecular markers for valuable traits used labor-intensive and sometimes poorly reproducible approaches. However, they allowed an assessment of the genetic diversity of flax and its relatives, the construction of linkage maps, and the identification of some markers for important characteristics. The sequencing of flax whole genome triggered the development of genome-wide association studies (GWAS) and quantitative trait locus (QTL) mapping. QTLs and quantitative trait nucleotides (QTNs) were identified for valuable seed- and fiber-related features and for resistance to biotic and abiotic stressors. Cost-effective and accurate analysis of large number of genotypes for multiple markers simultaneously using microarrays or targeted deep sequencing became available, as well as HRM, TaqMan, KASP, and other fluorescence-based high-throughput methods for detecting DNA polymorphisms. However, most DNA markers identified in flax are ambiguously linked to trait expression and are not universally applicable. A major challenge remains the lack of knowledge on functional polymorphisms. To date, only a few are known, mainly mutations in the FAD3 genes responsible for reduced linolenic acid content in linseed oil. For the further development of marker-assisted and genomic selection of flax, it is necessary to analyze exhaustively phenotyped sample sets, to identify DNA polymorphisms that determine valuable traits, and to develop efficient DNA test systems. [ABSTRACT FROM AUTHOR]

KAS II (β-ketoacyl-acyl carrier protein (ACP) synthases II), FAT (fatty acid thioesterases), SAD (stearoyl-ACP desaturase), and FAD (fatty acid desaturases) are the vital gene families involved in fatty acid (FA) synthesis in Hippophae rhamnoides L. However, information on the number and location of these genes and which ones are key to the formation of FAs in fruit seeds and pulp was not complete. Our study aimed to solve this issue using the available genomic sequences and transcriptome data that we obtained. We compared the protein sequences of sea buckthorn with those of Arabidopsis thaliana and checked for the presence of conserved domains. As a result of structure and phylogenetic analyses, 4 KAS II, 8 FAT, 9 SAD, and 12 FAD genes were identified in the H. rhamnoides genome, which were classified into subfamilies: KAS II, FATA, FATB, FAD2, FAD3, FAD6, and FAD7/8. To analyze the expression of the identified genes, we sequenced the transcriptomes of sea buckthorn seeds and fruit pulp at four development stages, as well as leaves. The analysis revealed representatives of the FAT, SAD, and FAD families with high tissue-and stage-specific expression in seeds and pulp. These genes are likely to play a key role in the biosynthesis of sea buckthorn FAs. The obtained results may help to establish the precise biosynthesis mechanisms of FAs and will promote the breeding of new sea buckthorn varieties that have oil with a defined FA composition. [ABSTRACT FROM AUTHOR]

Flax is an important crop grown for seed and fiber. Flax chromosome number is 2n = 30, and its genome size is about 450–480 Mb. To date, the genomes of several flax varieties have been sequenced and assembled. However, the obtained assemblies are still far from the telomere-to-telomere (T2T) level. We sequenced the genome of flax variety K-3018 on the Oxford Nanopore Technologies (ONT) platform and obtained 57.7 Gb of R10 simplex reads with an N50 = 18.4 kb (~120× genome coverage). ONT reads longer than 50 kb were kept as ultra-long ones (~10× genome coverage), and the rest of the ONT reads were corrected using the HERRO R10 model (quality > Q10, length > 10 kb, ~60× genome coverage remained). The genome was assembled using Hifiasm and Verkko. The Hifiasm-generated assembly was 489.1 Mb in length with 54 contigs and an N50 = 28.1 Mb. Verkko produced a very similar but more fragmented genome: 489.1 Mb, 134 contigs, N50 = 17.4 Mb. In the assembly by Hifiasm, eight chromosomes consisted of a single contig with telomeric repeats at both ends. In addition, five chromosomes comprised two contigs and two chromosomes comprised three contigs. These chromosomes also had telomeric repeats at their ends. The Hifiasm-generated assembly of variety K-3018 had similar contiguity but was likely more complete and accurate than the main fifteen-chromosome assembly of variety YY5 (produced from PacBio data and scaffolded with Hi-C data), the most contiguous flax genome assembly at the time of this writing. We suggest that sufficient genome coverage with long ONT R10 simplex reads is a viable alternative to PacBio plus Hi-C data for a high-precision T2T genome assembly of flax, opening new perspectives for whole-genome studies of flax. [ABSTRACT FROM AUTHOR]

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]

Flax is grown worldwide for seed and fiber production. Linseed varieties differ in their oil composition and are used in pharmaceutical, food, feed, and industrial production. The field of application primarily depends on the content of linolenic (LIN) and linoleic (LIO) fatty acids. Inactivating mutations in the FAD3A and FAD3B genes lead to a decrease in the LIN content and an increase in the LIO content. For the identification of the three most common low-LIN mutations in flax varieties (G-to-A in exon 1 of FAD3A substituting tryptophan with a stop codon, C-to-T in exon 5 of FAD3A leading to arginine to a stop codon substitution, and C-to-T in exon 2 of FAD3B resulting in histidine to tyrosine substitution), three approaches were proposed: (1) targeted deep sequencing, (2) high resolution melting (HRM) analysis, (3) cleaved amplified polymorphic sequences (CAPS) markers. They were tested on more than a thousand flax samples of various types and showed promising results. The proposed approaches can be used in marker-assisted selection to choose parent pairs for crosses, separate heterogeneous varieties into biotypes, and select genotypes with desired homozygous alleles of the FAD3A and FAD3B genes at the early stages of breeding for the effective development of varieties with a particular LIN and LIO content, as well as in basic studies of the molecular mechanisms of fatty acid synthesis in flax seeds to select genotypes adequate to the tasks. [ABSTRACT FROM AUTHOR]