Selection of Flax Genotypes for Pan-Genomic Studies by Sequencing Tagmentation-Based Transcriptome Libraries.

Flax (Linum usitatissimum L.) products are used in the food, pharmaceutical, textile, polymer, medical, and other industries. The creation of a pan-genome will be an important advance in flax research and breeding. The selection of flax genotypes that sufficiently cover the species diversity is a crucial step for the pan-genomic study. For this purpose, we have adapted a method based on Illumina sequencing of transcriptome libraries prepared using the Tn5 transposase (tagmentase). This approach reduces the cost of sample preparation compared to commercial kits and allows the generation of a large number of cDNA libraries in a short time. RNA-seq data were obtained for 192 flax plants (3-6 individual plants from 44 flax accessions of different morphology and geographical origin). Evaluation of the genetic relationship between flax plants based on the sequencing data revealed incorrect species identification for five accessions. Therefore, these accessions were excluded from the sample set for the pan-genomic study. For the remaining samples, typical genotypes were selected to provide the most comprehensive genetic diversity of flax for pan-genome construction. Thus, high-throughput sequencing of tagmentation-based transcriptome libraries showed high efficiency in assessing the genetic relationship of flax samples and allowed us to select genotypes for the flax pan-genomic analysis.

Assembling Quality Genomes of Flax Fungal Pathogens from Oxford Nanopore Technologies Data.

Flax (Linum usitatissimum L.) is attacked by numerous devastating fungal pathogens, including Colletotrichum lini, Aureobasidium pullulans, and Fusarium verticillioides (Fusarium moniliforme). The effective control of flax diseases follows the paradigm of extensive molecular research on pathogenicity. However, such studies require quality genome sequences of the studied organisms. This article reports on the approaches to assembling a high-quality fungal genome from the Oxford Nanopore Technologies data. We sequenced the genomes of C. lini, A. pullulans, and F. verticillioides (F. moniliforme) and received different volumes of sequencing data: 1.7 Gb, 3.9 Gb, and 11.1 Gb, respectively. To obtain the optimal genome sequences, we studied the effect of input data quality and genome coverage on assembly statistics and tested the performance of different assembling and polishing software. For C. lini, the most contiguous and complete assembly was obtained by the Flye assembler and the Homopolish polisher. The genome coverage had more effect than data quality on assembly statistics, likely due to the relatively low amount of sequencing data obtained for C. lini. The final assembly was 53.4 Mb long and 96.4% complete (according to the glomerellales_odb10 BUSCO dataset), consisted of 42 contigs, and had an N50 of 4.4 Mb. For A. pullulans and F. verticillioides (F. moniliforme), the best assemblies were produced by Canu-Medaka and Canu-Homopolish, respectively. The final assembly of A. pullulans had a length of 29.5 Mb, 99.4% completeness (dothideomycetes_odb10), an N50 of 2.4 Mb and consisted of 32 contigs. F. verticillioides (F. moniliforme) assembly was 44.1 Mb long, 97.8% complete (hypocreales_odb10), consisted of 54 contigs, and had an N50 of 4.4 Mb. The obtained results can serve as a guideline for assembling a de novo genome of a fungus. In addition, our data can be used in genomic studies of fungal pathogens or plant-pathogen interactions and assist in the management of flax diseases.

Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-Quality Genome.

High-quality genome sequences help to elucidate the genetic basis of numerous biological processes and track species evolution. For flax (Linum usitatissimum L.)-a multifunctional crop, high-quality assemblies from Oxford Nanopore Technologies (ONT) data were unavailable, largely due to the difficulty of isolating pure high-molecular-weight DNA. This article proposes a scheme for gaining a contiguous L. usitatissimum assembly using Nanopore data. We developed a protocol for flax nuclei isolation with subsequent DNA extraction, which allows obtaining about 5 µg of pure high-molecular-weight DNA from 0.5 g of leaves. Such an amount of material can be collected even from a single plant and yields more than 30 Gb of ONT data in two MinION runs. We performed a comparative analysis of different genome assemblers and polishers on the gained data and obtained the final 447.1-Mb assembly of L. usitatissimum line 3896 genome using the Canu-Racon (two iterations)-Medaka combination. The genome comprised 1695 contigs and had an N50 of 6.2 Mb and a completeness of 93.8% of BUSCOs from eudicots_odb10. Our study highlights the impact of the chosen genome construction strategy on the resulting assembly parameters and its eligibility for future genomic studies.

Development and Complex Application of Methods for the Identification of Mutations in the FAD3A and FAD3B Genes Resulting in the Reduced Content of Linolenic Acid in Flax Oil.

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.

Genes Associated with the Flax Plant Type (Oil or Fiber) Identified Based on Genome and Transcriptome Sequencing Data.

As a result of the breeding process, there are two main types of flax (Linum usitatissimum L.) plants. Linseed is used for obtaining seeds, while fiber flax is used for fiber production. We aimed to identify the genes associated with the flax plant type, which could be important for the formation of agronomically valuable traits. A search for polymorphisms was performed in genes involved in the biosynthesis of cell wall components, lignans, fatty acids, and ion transport based on genome sequencing data for 191 flax varieties. For 143 of the 424 studied genes (4CL, C3'H, C4H, CAD, CCR, CCoAOMT, COMT, F5H, HCT, PAL, CTL, BGAL, ABC, HMA, DIR, PLR, UGT, TUB, CESA, RGL, FAD, SAD, and ACT families), one or more polymorphisms had a strong correlation with the flax type. Based on the transcriptome sequencing data, we evaluated the expression levels for each flax type-associated gene in a wide range of tissues and suggested genes that are important for the formation of linseed or fiber flax traits. Such genes were probably subjected to the selection press and can determine not only the traits of seeds and stems but also the characteristics of the root system or resistance to stresses at a particular stage of development, which indirectly affects the ability of flax plants to produce seeds or fiber.

Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.

Aluminum Responsive Genes in Flax (Linum usitatissimum L.).

Flax (Linum usitatissimum L.) is a multipurpose crop which is used for the production of textile, oils, composite materials, pharmaceuticals, etc. Soil acidity results in a loss of seed and fiber production of flax, and aluminum toxicity is a major factor that depresses plant growth and development in acid conditions. In the present work, we evaluated gene expression alterations in four flax genotypes with diverse tolerance to aluminum exposure. Using RNA-Seq approach, we revealed genes that are differentially expressed under aluminum stress in resistant (Hermes, TMP1919) and sensitive (Lira, Orshanskiy) cultivars and selectively confirmed the identified alterations using qPCR. To search for differences in response to aluminum between resistant and sensitive genotypes, we developed the scoring that allowed us to suggest the involvement of MADS-box and NAC transcription factors regulating plant growth and development and enzymes participating in cell wall modifications in aluminum tolerance in flax. Using Gene Ontology (GO) enrichment analysis, we revealed that glutathione metabolism, oxidoreductase, and transmembrane transporter activities are the most affected by the studied stress in flax. Thus, we identified genes that are involved in aluminum response in resistant and sensitive genotypes and suggested genes that contribute to flax tolerance to the aluminum stress.