Flax (Linum usitatissimum L.) is a crop plant used for fiber and oil production. Although potentially high-yielding flax varieties have been developed, environmental stresses markedly decrease flax production. Among biotic stresses, Fusarium oxysporum f. sp. lini is recognized as one of the most devastating flax pathogens. It causes wilt disease that is one of the major limiting factors for flax production worldwide. Breeding and cultivation of flax varieties resistant to F. oxysporum is the most effective method for controlling wilt disease. Although the mechanisms of flax response to Fusarium have been actively studied, data on the plant response to infection and resistance gene candidates are currently very limited. The transcriptomes of two resistant and two susceptible flax cultivars with respect to Fusarium wilt, as well as two resistant BC2F5 populations, which were grown under control conditions or inoculated with F. oxysporum, were sequenced using the Illumina platform. Genes showing changes in expression under F. oxysporum infection were identified in both resistant and susceptible flax genotypes. We observed the predominant overexpression of numerous genes that are involved in defense response. This was more pronounced in resistant cultivars. In susceptible cultivars, significant downregulation of genes involved in cell wall organization or biogenesis was observed in response to F. oxysporum. In the resistant genotypes, upregulation of genes related to NAD(P)H oxidase activity was detected. Upregulation of a number of genes, including that encoding beta-1,3-glucanase, was significantly greater in the cultivars and BC2F5 populations resistant to Fusarium wilt than in susceptible cultivars in response to F. oxysporum infection. Using high-throughput sequencing, we identified genes involved in the early defense response of L. usitatissimum against the fungus F. oxysporum. In response to F. oxysporum infection, we detected changes in the expression of pathogenesis-related protein-encoding genes and genes involved in ROS production or related to cell wall biogenesis. Furthermore, we identified genes that were upregulated specifically in flax genotypes resistant to Fusarium wilt. We suggest that the identified genes in resistant cultivars and BC2F5 populations showing induced expression in response to F. oxysporum infection are the most promising resistance gene candidates.

Cultivated flax (Linum usitatissimum L.) is widely used for production of textile, food, chemical and pharmaceutical products. However, various stresses decrease flax production. Search for genes, which are involved in stress response, is necessary for breeding of adaptive cultivars. Imbalanced concentration of nutrient elements in soil decrease flax yields and also results in heritable changes in some flax lines. The appearance of Linum Insertion Sequence 1 (LIS-1) is the most studied modification. However, LIS-1 function is still unclear. High-throughput sequencing of transcriptome of flax plants grown under normal (N), phosphate deficient (P), and nutrient excess (NPK) conditions was carried out using Illumina platform. The assembly of transcriptome was performed, and a total of 34924, 33797, and 33698 unique transcripts for N, P, and NPK sequencing libraries were identified, respectively. We have not revealed any LIS-1 derived mRNA in our sequencing data. The analysis of high-throughput sequencing data allowed us to identify genes with potentially differential expression under imbalanced nutrition. For further investigation with qPCR, 15 genes were chosen and their expression levels were evaluated in the extended sampling of 31 flax plants. Significant expression alterations were revealed for genes encoding WRKY and JAZ protein families under P and NPK conditions. Moreover, the alterations of WRKY family genes differed depending on LIS-1 presence in flax plant genome. Besides, we revealed slight and LIS-1 independent mRNA level changes of KRP2 and ING1 genes, which are adjacent to LIS-1, under nutrition stress. Differentially expressed genes were identified in flax plants, which were grown under phosphate deficiency and excess nutrition, on the basis of high-throughput sequencing and qPCR data. We showed that WRKY and JAS gene families participate in flax response to imbalanced nutrient content in soil. Besides, we have not identified any mRNA, which could be derived from LIS-1, in our transcriptome sequencing data. Expression of LIS-1 flanking genes, ING1 and KRP2, was suggested not to be nutrient stress-induced. Obtained results provide new insights into edaphic stress response in flax and the role of LIS-1 in these process.

About 30% of the world's ice-free land area is occupied by acid soils. In soils with pH below 5, aluminum (Al) releases to the soil solution and becomes highly toxic for plants. Therefore, breeding of varieties that are resistant to Al is needed. Flax (Linum usitatissimum L.) is grown worldwide for fiber and seed production. Al toxicity in acid soils is a serious problem for flax cultivation. However, very little is known about mechanisms of flax resistance to Al and the genetics of this resistance. In the present work, we sequenced 16 transcriptomes of flax cultivars resistant (Hermes and TMP1919) and sensitive (Lira and Orshanskiy) to Al, which were exposed to control conditions and aluminum treatment for 4, 12, and 24 hours. In total, 44.9–63.3 million paired-end 100-nucleotide reads were generated for each sequencing library. Based on the obtained high-throughput sequencing data, genes with differential expression under aluminum exposure were revealed in flax. The majority of the top 50 up-regulated genes were involved in transmembrane transport and transporter activity in both the Al-resistant and Al-sensitive cultivars. However, genes encoding proteins with glutathione transferase and UDP-glycosyltransferase activity were in the top 50 up-regulated genes only in the flax cultivars resistant to aluminum. For qPCR analysis in extended sampling, two UDP-glycosyltransferases (UGTs) and three glutathione S-transferases (GSTs) were selected. The general trend of alterations in the expression of the examined genes was the up-regulation under Al stress, especially after 4 h of Al exposure. Moreover, in the flax cultivars resistant to aluminum, the increase in expression was more pronounced than that in the sensitive cultivars. We speculate that the defense against the Al toxicity via GST antioxidant activity is the probable mechanism of the response of flax plants to aluminum stress. We also suggest that UGTs could be involved in cell wall modification and protection from reactive oxygen species (ROS) in response to Al stress in L. usitatissimum. Thus, GSTs and UGTs, probably, play an important role in the response of flax to Al via detoxification of ROS and cell wall modification.

The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n = 30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes. High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH. Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n = 30) could originate either as the result of hybridization of two diploid species (2n = 16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n = 16) and a diploid ancestor of modern L. narbonense (2n = 14). High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.

Acid soils limit agricultural production worldwide. Major reason of crop losses in acid soils is the toxicity of aluminum (Al). In the present work, we investigated expression alterations of microRNAs in flax (Linum usitatissimum L.) plants under Al stress. Flax seedlings of resistant (TMP1919 and G1071/4_k) and sensitive (Lira and G1071/4_o) to Al cultivars and lines were exposed to AlCl₃ solution for 4 and 24 hours. Twelve small RNA libraries were constructed and sequenced using Illumina platform. In total, 97 microRNAs from 18 conserved families were identified. miR319, miR390, and miR393 revealed expression alterations associated with Al treatment of flax plants. Moreover, for miR390 and miR393, the alterations were distinct in sensitive and resistant to Al genotypes. Expression level changes of miR319 and miR390 were confirmed using qPCR analysis. In flax, potential targets of miR319 are TCPs, miR390–TAS3 and GRF5, and miR393–AFB2-coding transcripts. TCPs, TAS3, GRF5, and AFB2 participate in regulation of plant growth and development. The involvement of miR319, miR390, and miR393 in response to Al stress in flax was shown here for the first time. We speculate that these microRNAs play an important role in Al response via regulation of growth processes in flax plants.