Key FAD2, FAD3, and SAD Genes Involved in the Fatty Acid Synthesis in Flax Identified Based on Genomic and Transcriptomic Data

FAD (fatty acid desaturase) and SAD (stearoyl-ACP desaturase) genes play key roles in the synthesis of fatty acids (FA) and determination of oil composition in flax (Linum usitatissimum L.). We searched for FAD and SAD genes in the most widely used flax genome of the variety CDC Bethune and three available long-read assembled flax genomes—YY5, 3896, and Atlant. We identified fifteen FAD2, six FAD3, and four SAD genes. Of all the identified genes, 24 were present in duplicated pairs. In most cases, two genes from a pair differed by a significant number of gene-specific SNPs (single nucleotide polymorphisms) or even InDels (insertions/deletions), except for FAD2a-1 and FAD2a-2, where only seven SNPs distinguished these genes. Errors were detected in the FAD2a-1, FAD2a-2, FAD3c-1, and FAD3d-2 sequences in the CDC Bethune genome assembly but not in the long-read genome assemblies. Expression analysis of the available transcriptomic data for different flax organs/tissues revealed that FAD2a-1, FAD2a-2, FAD3a, FAD3b, SAD3-1, and SAD3-2 were specifically expressed in embryos/seeds/capsules and could play a crucial role in the synthesis of FA in flax seeds. In contrast, FAD2b-1, FAD2b-2, SAD2-1, and SAD2-2 were highly expressed in all analyzed organs/tissues and could be involved in FA synthesis in whole flax plants. FAD2c-2, FAD2d-1, FAD3c-1, FAD3c-2, FAD3d-1, FAD3d-2, SAD3-1, and SAD3-2 showed differential expression under stress conditions—Fusarium oxysporum infection and drought. The obtained results are essential for research on molecular mechanisms of fatty acid synthesis, FAD and SAD editing, and marker-assisted and genomic selection for breeding flax varieties with a determined fatty acid composition of oil.

In the work by Thambugala et al., SAD and FAD loci were compared between the high-LIN cultivar CDC Bethune and the low-LIN line M5791 using bacterial artificial chromosome clones.This allowed researchers to gain insight into the structural organization and diversity of these regions of the flax genome [51].
In the past few years, the genome assembly of CDC Bethune has been significantly improved with different techniques, including optical mapping [52].Moreover, several genome assemblies of other flax genotypes were obtained from the third-generation sequencing and/or Hi-C data [53][54][55][56].Long reads allow reliable assembly of highly homologous contiguous loci and enable the evaluation of the differences between pairs of duplicated genes [57,58].The obtained flax genomes opened up new opportunities for the identification and comparison of the SAD, FAD2, and FAD3 genes of different flax genotypes.In addition to whole-genome sequencing, a significant number of transcriptomes have been obtained for flax plants in recent years [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75].Such studies enabled extensive analysis of SAD, FAD2, and FAD3 expression in different organs/tissues and during various developmental stages and under various stress conditions.The obtained data are necessary to determine the role of SAD and FAD in FA synthesis and other processes in flax plants.
Our work aimed at the genome-wide identification of FAD2, FAD3, and SAD genes in the most frequently used CDC Bethune genome and three flax genomes sequenced on third-generation platforms.We also analyzed the expression of these genes in different flax organs/tissues and under different stress conditions.

Identification of FAD2 Genes in Flax Genome Assemblies
Using sequences of the FAD2 genes from the study of You et al. [46], we identified the location of the FAD2 genes in four analyzed genomes: fiber flax YY5 (Yiya No. 5; from PacBio HiFi and Hi-C data [54]); linseed line 3896 (from Nanopore data [56]); fiber flax Atlant (from Nanopore data [55]); and linseed CDC Bethune (from Illumina and optical mapping data [52]).Gene coordinates are presented in Table 1.Fifteen FAD2 genes were identified in each studied genome, except for that of cultivar Atlant.In this genotype, additional FAD2b-2* and FAD2c-1* genes were revealed.These copies were localized in one contig (JACHUY010001688.1).Several additional SNPs (single nucleotide polymorphisms) and short InDels (insertions/deletions) were revealed in FAD2c-1* compared to other FAD2c genes identified in the studied flax varieties.The Atlant genome assembly is less contiguous than two other long-read flax genome assemblies (N50 = 0.4 Mb for Atlant, N50 = 6.2 Mb for 3896, and N50 = 9.6 Mb for YY5) [54][55][56].Therefore, the presence of two additional FAD2 genes is unlikely to be the Atlant genotype feature but could be associated with errors in the genome assembly.To avoid inaccuracies, we excluded the FAD2b-2* and FAD2c-1* genes of Atlant from further analysis.

Phylogenetic Analysis of FAD2 Genes of Different Flax Varieties
We analyzed sequences of 60 FAD2 genes that we identified in four flax varieties (15 genes per variety) (sequences are presented in Supplementary Table S1).A dendrogram for the FAD2 gene sequences is presented in Figure 1.The sequences of the same FAD2 genes of different varieties clustered with each other and formed subclusters.However, two genes were exceptions: FAD2a-1 and FAD2a-2 of CDC Bethune.For these genes, You et al. showed the presence of mis-assemblies in the previous version of the CDC Bethune genome (https://phytozome-next.jgi.doe.gov/info/Lusitatissimum_v1_0,accessed on 1 August 2023) [46].Probably, the current version of the CDC Bethune genome assembly (NCBI GenBank, GCA_000224295.2) can still have errors in the FAD2a-1 and FAD2a-2 sequences.Indeed, we found a long deletion in FAD2a-1 and an insertion in FAD2a-2 of CDC Bethune compared to these genes of varieties YY5, 3896, and Atlant.In the YY5, 3896, and Atlant genomes, the FAD2a-1 gene sequences were identical; the same was true for the FAD2a-2 gene.FAD2a-1 differed from FAD2a-2 by only seven SNPs in all three varieties.Thus, FAD2a-1 and FAD2a-2 are very conserved in flax and can be distinguished one from another by seven SNPs.Notably, one part of the FAD2a-1 gene of CDC Bethune was similar to the FAD2a-2 gene of YY5, 3896, and Atlant, but not FAD2a-1 of these varieties.This also indicated an error in the FAD2a-1 sequence of CDC Bethune.
CDC Bethune compared to these genes of varieties YY5, 3896, and Atlant.In the YY5, 3896, and Atlant genomes, the FAD2a-1 gene sequences were identical; the same was true for the FAD2a-2 gene.FAD2a-1 differed from FAD2a-2 by only seven SNPs in all three varieties.Thus, FAD2a-1 and FAD2a-2 are very conserved in flax and can be distinguished one from another by seven SNPs.Notably, one part of the FAD2a-1 gene of CDC Bethune was similar to the FAD2a-2 gene of YY5, 3896, and Atlant, but not FAD2a-1 of these varieties.This also indicated an error in the FAD2a-1 sequence of CDC Bethune.Generally, all pairs of the duplicated FAD2 genes differed from each other by a large number of SNPs and several InDels.The analysis of the FAD2 sequences of the four flax varieties allowed us to separate polymorphisms that were specific to a particular FAD2 gene from those specific to a genotype.In most pairs of the duplicated FAD2 genes (FAD2b-1/FAD2b-2, FAD2c-1/FAD2c-2, FAD2d-1/FAD2d-2, FAD2e-1/FAD2e-2, FAD2f-1/FAD2f-2, FAD2g-1/FAD2g-2), we revealed a significant number of SNPs that were present in all studied flax varieties and allowed distinguishing between two genes from a pair.The FAD2a-1/FAD2a-2 pair was an exception, where FAD2a-1 differed from FAD2a-2 by only seven SNPs.

Identification and Phylogenetic Analysis of FAD3 Genes in Genomes of Different Flax Varieties
Using the sequences of the FAD3a, FAD3b, FAD3c-1, FAD3c-2, FAD3d-1, and FAD3d-2 flax genes described by You et al. [46], we identified the FAD3 genes in the genomes of the flax varieties YY5, 3896, Atlant, and CDC Bethune.Gene coordinates are presented in Table 3.We analyzed the sequences of the FAD3a, FAD3b, FAD3c-1, FAD3c-2, FAD3d-1, and FAD3d-2 genes of the four flax varieties (sequences are presented in Supplementary Table S1).A dendrogram for the FAD3 gene sequences is presented in Figure 2. The sequences of the same FAD3 genes of different flax varieties clustered with each other and formed subclusters.Subclusters of the duplicated genes grouped together: FAD3a and FAD3b, FAD3c-1 and FAD3c-2, FAD3d-1 and FAD3d-2.Pairs of the duplicated FAD3 genes differed from each other by a significant number of SNPs and InDels.For each pair of the duplicated FAD3 genes (FAD3a/FAD3b, FAD3c-1/FAD3c-2, and FAD3d-1/FAD3d-2), we revealed a significant number of SNPs and several short InDels that were present in all studied flax varieties and allowed distinguishing between two genes from a pair.We observed the similarity between the sequences of the same FAD3 genes of the studied genotypes: few SNPs/InDels were revealed for FAD3c-2, FAD3d-1, FAD3a, and FAD3b.The FAD3d-2 gene had no polymorphisms in the YY5, 3896, or Atlant genomes.However, this gene of CDC Bethune had about a 240-bp deletion compared to FAD3d-2 of the other three varieties.The sequence before the deletion corresponded to FAD3d-1 of YY5, 3896, and Atlant, while the sequence after the deletion corresponded to FAD3d-2 of these varieties.We suggested that this was rather an error in the CDC Bethune genome assembly than a genotype feature.A similar picture was observed for FAD3c-1-one region of several dozens of nucleotides in length contained SNPs and InDels and was present only in FAD3c-1 of CDC Bethune.In the same region of FAD3c-1 in the three long-read flax genome assemblies, there was a slight difference in the length of homopolymers.However, this could be an assembly error.Similar to the analysis of the FAD2 genes, the analysis of the FAD3 sequences of the four flax varieties allowed us to distinguish polymorphisms that were specific to a particular FAD3 gene from those specific to a genotype.
varieties.The sequence before the deletion corresponded to FAD3d-1 of YY5, 3896, and Atlant, while the sequence after the deletion corresponded to FAD3d-2 of these varieties.We suggested that this was rather an error in the CDC Bethune genome assembly than a genotype feature.A similar picture was observed for FAD3c-1-one region of several dozens of nucleotides in length contained SNPs and InDels and was present only in FAD3c-1 of CDC Bethune.In the same region of FAD3c-1 in the three long-read flax genome assemblies, there was a slight difference in the length of homopolymers.However, this could be an assembly error.Similar to the analysis of the FAD2 genes, the analysis of the FAD3 sequences of the four flax varieties allowed us to distinguish polymorphisms that were specific to a particular FAD3 gene from those specific to a genotype.

Identification and Phylogenetic Analysis of SAD Genes in Genomes of Different Flax Varieties
Using the sequences of the SAD2-1, SAD2-2, SAD3-1, and SAD3-2 flax genes described by You et al. [46], we identified the SAD genes in the genomes of flax varieties YY5, 3896, Atlant, and CDC Bethune.Gene coordinates are presented in Table 4.
We compared the SAD2-1, SAD2-2, SAD3-1, and SAD3-2 gene sequences of the four flax varieties (sequences are presented in Supplementary Table S1).A dendrogram for the SAD gene sequences is presented in Figure 3.The sequences of the same SAD genes of different flax varieties clustered with each other and formed subclusters.SAD2-1 and SAD2-2 subclusters, as well as SAD3-1 and SAD3-2 subclusters, grouped together.The SAD2-1/SAD2-2 pair differed from the SAD3-1/SAD3-2 pair by a significant number of SNPs and InDels.For each pair of the duplicated SAD genes, we revealed a significant number of SNPs and several short InDels that were present in all the studied flax varieties and allowed distinguishing between two genes from a pair.The sequences of the same SAD genes were similar enough in the studied genotypes and mostly differed by single

Identification and Phylogenetic Analysis of SAD Genes in Genomes of Different Flax Varieties
Using the sequences of the SAD2-1, SAD2-2, SAD3-1, and SAD3-2 flax genes described by You et al. [46], we identified the SAD genes in the genomes of flax varieties YY5, 3896, Atlant, and CDC Bethune.Gene coordinates are presented in Table 4.We compared the SAD2-1, SAD2-2, SAD3-1, and SAD3-2 gene sequences of the four flax varieties (sequences are presented in Supplementary Table S1).A dendrogram for the SAD gene sequences is presented in Figure 3.The sequences of the same SAD genes of different flax varieties clustered with each other and formed subclusters.SAD2-1 and SAD2-2 subclusters, as well as SAD3-1 and SAD3-2 subclusters, grouped together.The SAD2-1/SAD2-2 pair differed from the SAD3-1/SAD3-2 pair by a significant number of SNPs and InDels.For each pair of the duplicated SAD genes, we revealed a significant number of SNPs and several short InDels that were present in all the studied flax varieties and allowed distinguishing between two genes from a pair.The sequences of the same SAD genes were similar enough in the studied genotypes and mostly differed by single SNPs.However, a 20-30-nucleotide InDel and several 1-5-nucleotide InDels were identified in the SAD3-1 gene.Two one-nucleotide InDels were revealed in the SAD3-2 gene within the studied varieties.Thus, the analysis of the SAD sequences of the four flax varieties enabled distinguishing between polymorphisms that were specific to a particular SAD gene and those specific to a genotype.
fied in the SAD3-1 gene.Two one-nucleotide InDels were revealed in the SAD3-2 gene within the studied varieties.Thus, the analysis of the SAD sequences of the four flax varieties enabled distinguishing between polymorphisms that were specific to a particular SAD gene and those specific to a genotype.

Expression Analysis of FAD2 Genes
We analyzed the expression of the FAD2 genes in different flax organs/tissues and under various biotic/abiotic stress conditions (Figures 4 and 5, Supplementary Table S2).The highest expression levels were observed for FAD2b-1 and FAD2b-2 (Figure 4a).These genes were expressed at high levels in all the analyzed samples.For the other FAD2 genes, expression was more organ-/tissue-specific.Notably, FAD2a-1 and FAD2a-2 were predominantly expressed in embryos, endosperm, seeds, and capsules, suggesting their significant role in the synthesis of flax oil FAs (Figure 4b).Increased expression of FAD2a-1

Expression Analysis of FAD2 Genes
We analyzed the expression of the FAD2 genes in different flax organs/tissues and under various biotic/abiotic stress conditions (Figures 4 and 5, Supplementary Table S2).The highest expression levels were observed for FAD2b-1 and FAD2b-2 (Figure 4a).These genes were expressed at high levels in all the analyzed samples.For the other FAD2 genes, expression was more organ-/tissue-specific.Notably, FAD2a-1 and FAD2a-2 were predominantly expressed in embryos, endosperm, seeds, and capsules, suggesting their significant role in the synthesis of flax oil FAs (Figure 4b).Increased expression of FAD2a-1 was also observed in leaves in one of the used transcriptomic datasets (Figure 4b).Among the FAD2 genes, FAD2b-1 and FAD2b-2 had the highest expression levels in embryos, endosperm, seeds, and capsules (Figure 4b).However, the expression of FAD2b-1 and FAD2b-2 was also high in other studied organs/tissues (Figure 4b).Therefore, these genes could play a key role in common processes in all flax organs and tissues, as well as the synthesis of flax oil FAs.Pronounced organ-/tissue-specific expression was observed for the FAD2c-1, FAD2d-1, and FAD2f-1 genes.FAD2c-1 had an increased expression level in the xylem part of stem, compared to phloem fibers, the apical part, and the parenchyma of stem (Figure 4b).Probably, this gene is involved in the functioning of stem xylem tissues.For FAD2d-1 and FAD2f-1, high expression levels were specific to root samples (Figures 4b and 5b).Therefore, these genes could have a significant role in root cells.
the FAD2 genes, FAD2b-1 and FAD2b-2 had the highest expression levels in embryos, endosperm, seeds, and capsules (Figure 4b).However, the expression of FAD2b-1 and FAD2b-2 was also high in other studied organs/tissues (Figure 4b).Therefore, these genes could play a key role in common processes in all flax organs and tissues, as well as the synthesis of flax oil FAs.Pronounced organ-/tissue-specific expression was observed for the FAD2c-1, FAD2d-1, and FAD2f-1 genes.FAD2c-1 had an increased expression level in the xylem part of stem, compared to phloem fibers, the apical part, and the parenchyma of stem (Figure 4b).Probably, this gene is involved in the functioning of stem xylem tissues.For FAD2d-1 and FAD2f-1, high expression levels were specific to root samples (Figures 4b and 5b).Therefore, these genes could have a significant role in root cells.S2), so the color scale in Figures 4 and 5 S2), so the color scale in Figures 4 and 5  Along with organ-/tissue-specific expression, we discovered the effects of different stressors on FAD2 expression in the analyzed flax samples.As such, Fusarium oxysporum inoculation increased FAD2c-2 expression in roots (Figure 5b).This effect was most prominent in the data from the study of the early response to the fungus (NCBI BioProject, PRJNA412801).The other experimental data (PRJNA432224) demonstrated that the increased expression of FAD2c-2 was more associated with F. oxysporum than other fungal species (Supplementary Table S2).For FAD2d-1, differential expression was detected in the samples from the study of drought influence on flax.The increased expression was characteristic of samples under repeated drought stress and re-watering conditions, compared to control and drought conditions (Figure 5b).However, no FAD2 genes were activated in response to the other analyzed abiotic stresses (different soil pH, increased concentration of aluminum (Al), and zinc (Zn) deficiency) (Figure 5b).
stressors on FAD2 expression in the analyzed flax samples.As such, Fusarium oxysporum inoculation increased FAD2c-2 expression in roots (Figure 5b).This effect was most prominent in the data from the study of the early response to the fungus (NCBI BioProject, PRJNA412801).The other experimental data (PRJNA432224) demonstrated that the increased expression of FAD2c-2 was more associated with F. oxysporum than other fungal species (Supplementary Table S2).For FAD2d-1, differential expression was detected in the samples from the study of drought influence on flax.The increased expression was characteristic of samples under repeated drought stress and re-watering conditions, compared to control and drought conditions (Figure 5b).However, no FAD2 genes were activated in response to the other analyzed abiotic stresses (different soil pH, increased concentration of aluminum (Al), and zinc (Zn) deficiency) (Figure 5b).S2), so the color scale in Figures 4 and 5 is common.Noted gene expression patterns of interest are highlighted in boxes.

Expression Analysis of FAD3 Genes
FAD3 expression was analyzed for different flax organs/tissues and biotic/abiotic stress conditions (Figures 6 and 7, Supplementary Table S2).FAD3a and FAD3b were expressed at high levels in flowers, capsules, and seeds (Figure 6a), with the most remarkable expression increase at the torpedo and cotyledon stages of embryo development (Supplementary Table S2).However, the expression was either low or zero in the majority of other tissues (Figure 6a).FAD3c-1 and FAD3c-2 had increased expression in most root tissues (PRJNA634481, PRJNA497472, PRJNA432224, PRJNA412801); however, they were also expressed in other organs/tissues (Figures 6b and 7b).Meanwhile, decreased expression of FAD3c-1 and FAD3c-2 was observed for samples inoculated with F. oxysporum (PRJNA412801) (Figure 7b).Therefore, these genes could be involved in the response to the pathogen.In contrast to FAD3c-1 and FAD3c-2, inoculation with F. oxysporum increased the expression of FAD3d-1 and FAD3d-2 (PRJNA412801) (Figure 7b).However, the expression of these genes decreased in flax samples under repeated drought conditions in the study of flax response to drought (Figure 7b).
ditions were analyzed together (as in Supplementary Table S2), so the color scale in Figures 4 and 5 is common.Noted gene expression patterns of interest are highlighted in boxes.

Expression Analysis of FAD3 Genes
FAD3 expression was analyzed for different flax organs/tissues and biotic/abiotic stress conditions (Figures 6 and 7, Supplementary Table S2).FAD3a and FAD3b were expressed at high levels in flowers, capsules, and seeds (Figure 6a), with the most remarkable expression increase at the torpedo and cotyledon stages of embryo development (Supplementary Table S2).However, the expression was either low or zero in the majority of other tissues (Figure 6a).FAD3c-1 and FAD3c-2 had increased expression in most root tissues (PRJNA634481, PRJNA497472, PRJNA432224, PRJNA412801); however, they were also expressed in other organs/tissues (Figures 6b and 7b).Meanwhile, decreased expression of FAD3c-1 and FAD3c-2 was observed for samples inoculated with F. oxysporum (PRJNA412801) (Figure 7b).Therefore, these genes could be involved in the response to the pathogen.In contrast to FAD3c-1 and FAD3c-2, inoculation with F. oxysporum increased the expression of FAD3d-1 and FAD3d-2 (PRJNA412801) (Figure 7b).However, the expression of these genes decreased in flax samples under repeated drought conditions in the study of flax response to drought (Figure 7b).S2), so the color scale in Figures 6 and 7 is common.Noted gene expression patterns of interest are highlighted in boxes.

Expression Analysis of SAD Genes
We analyzed the expression of the SAD genes in different flax organs/tissues and under various biotic/abiotic stress conditions (Figures 8 and 9, Supplementary Table S2).SAD2-1 had expression profiles close to those of SAD2-2.Similarly, the SAD3-1 expression profiles resembled those of SAD3-2 (Figures 8 and 9).
Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary Table S2), so the color scale in Figures 6 and 7 S2), so the color scale in Figures 6 and 7 is common.Noted gene expression patterns of interest are highlighted in boxes.

Expression Analysis of SAD Genes
We analyzed the expression of the SAD genes in different flax organs/tissues and under various biotic/abiotic stress conditions (Figures 8 and 9, Supplementary Table S2).SAD2-1 had expression profiles close to those of SAD2-2.Similarly, the SAD3-1 expression profiles resembled those of SAD3-2 (Figures 8 and 9).
For all SAD genes (SAD2-1, SAD2-2, SAD3-1, and SAD3-2), increased expression was characteristic of embryos, endosperm, and capsules (Figure 8a).However, SAD3-1 and SAD3-2 had an increased expression level in capsules only at the early development stages (PRJNA539945) (Supplementary Table S2).For SAD2-1 and SAD2-2, there were no significant differences between the various stages of capsule development.Notably, the difference between the expression levels of SAD2 and SAD3 was observed in the tissues of the developing flax embryo.While SAD2-1 and SAD2-2 had high expression levels in mature embryos, high SAD3-1 and SAD3-2 expression levels were characteristic of the early stages of embryo development (Supplementary Table S2).Therefore, SAD3-1 and SAD3-2 could be more important for the early stages of seed development.Meanwhile, SAD2-1 and SAD2-2 could also function at later stages of seed development.S2), so the color scale in Figures 6 and 7 is common.Noted gene expression patterns of interest are highlighted in boxes.S2), so the color scale in Figures 8 and 9 S2), so the color scale in Figures 8 and 9 S2), so the color scale in Figures 8 and 9 is common.Noted gene expression patterns of interest are highlighted in boxes.S2), so the color scale in Figures 8 and 9 is common.Noted gene expression patterns of interest are highlighted in boxes.
SAD2-1 and SAD2-2 were expressed in most analyzed flax organs/tissues (Figure 8a).Unlike SAD2-1 and SAD2-2, differences in the expression levels of SAD3-1 and SAD3-2 between organs/tissues were more pronounced (Figure 8a).In addition to embryos, endosperm, and capsules, SAD3-1 and SAD3-2 had increased expression levels in roots in most  S2), so the color scale in Figures 8 and 9 is common.Noted gene expression patterns of interest are highlighted in boxes.
For all SAD genes (SAD2-1, SAD2-2, SAD3-1, and SAD3-2), increased expression was characteristic of embryos, endosperm, and capsules (Figure 8a).However, SAD3-1 and SAD3-2 had an increased expression level in capsules only at the early development stages (PRJNA539945) (Supplementary Table S2).For SAD2-1 and SAD2-2, there were no significant differences between the various stages of capsule development.Notably, the difference between the expression levels of SAD2 and SAD3 was observed in the tissues of the developing flax embryo.While SAD2-1 and SAD2-2 had high expression levels in mature embryos, high SAD3-1 and SAD3-2 expression levels were characteristic of the early stages of embryo development (Supplementary Table S2).Therefore, SAD3-1 and SAD3-2 could be more important for the early stages of seed development.Meanwhile, SAD2-1 and SAD2-2 could also function at later stages of seed development.
SAD2-1 and SAD2-2 were expressed in most analyzed flax organs/tissues (Figure 8a).Unlike SAD2-1 and SAD2-2, differences in the expression levels of SAD3-1 and SAD3-2 between organs/tissues were more pronounced (Figure 8a).In addition to embryos, endosperm, and capsules, SAD3-1 and SAD3-2 had increased expression levels in roots in most studied experiments (all samples of PRJNA497472 and PRJNA412801 and most samples of PRJNA432224 and PRJNA634481) (Figure 8b).In the majority of the other organs/tissues, SAD3-1 and SAD3-2 were unexpressed or expressed at very low levels (Figure 8b).Possibly, the SAD3-1 and SAD3-2 genes are involved in some processes in roots but not in common processes in all flax organs/tissues.
In addition, we revealed trends in the changes of SAD expression in flax under stress conditions.In the study of drought effects, flax genotypes under control and drought conditions significantly differed in SAD2-1 and SAD2-2 expression (Figure 9b, Supplementary Table S2).However, genotype differences almost vanished under repeated drought stress and re-watering conditions (Figure 9b, Supplementary Table S2).For SAD3-1 and SAD3-2, increased expression was revealed in flax samples inoculated with F. oxysporum (Figure 9b).
However, the effective application of genome editing and RNA interference (RNAi) for the suppression of gene expression requires knowledge of the number and sequences of FAD2.The correct choice of genome editing targets also needs information on the differences between the FAD2 sequences and their expression levels in different tissues.In flax seeds/capsules, the genes of the OLE-LIO transformation are the most promising targets for creating flax varieties with high OLE content.Our analysis established that FAD2a-1 and FAD2a-2 were predominantly expressed in seeds/capsules, but FAD2b-1 and FAD2b-2 had higher expression in the same tissues.Moreover, FAD2b-1 and FAD2b-2 were expressed at high levels in all the studied flax organs/tissues.Another study showed that FAD2b had greater desaturase activity compared to that of FAD2a [48].Thus, creating flax varieties with high OLE content can be challenging.Probably, all four FAD genes (FAD2a-1/FAD2a-2 and FAD2b-1/FAD2b-2) should be inactivated.Meanwhile, the expression of FAD2b-1 and FAD2b-2 in all the studied flax organs/tissues could point out their vital role in the synthesis of FAs not only in plant seeds, where Fas serve as energy reserves, but also in all other organs/tissues, taking part in plant metabolism.Therefore, FAD2b-1 and FAD2b-2 inactivation could lead to a reduced capacity for flax survival.For example, manipulating the FAD2 genes could result in poor agronomic characteristics of the obtained high-OLE plants [1].However, RNAi-mediated silencing of the FAD2 genes in flax (the experiment was conducted to suppress FAD2a and FAD2b expression regardless of the gene duplicates) allowed obtaining high-OLE plants with a normal phenotype [33].In light of such studies, further development of high-OLE flax varieties is promising.
You et al. [46] analyzed the expression of the duplicated FAD and SAD genes only in pairs (except for FAD3a and FAD3b, which were treated individually) and in a small number of tissues.We demonstrated that genes from each pair differed by a significant number of SNPs and InDels (except for the FAD2a-1/FAD2a-2 pair, where only seven SNPs distinguished these genes).Due to these differences, we could analyze the expression of individual genes.Moreover, sequencing a great number of transcriptomes of different organs/tissues of flax plants (including those under biotic/abiotic stress conditions) enabled the analysis of FAD and SAD expression for a representative set of samples [59][60][61][62][63][64][65][66][67][68][69][70].
In this study, we revealed seed-/capsule-specific expression of FAD2a-1, FAD2a-2, FAD3a, FAD3b, SAD3-1, and SAD3-2.Therefore, these genes could substantially contribute to the determination of the FA composition of flax oil.Since oil composition determines the further application of flax seeds, it is one of the most important characteristics of linseed.FAD2b-1, FAD2b-2, SAD2-1, and SAD2-2 had high expression in embryos/seeds/capsules, as well as other tissues.Hence, these genes could be responsible for the synthesis of FAs in seeds and other flax organs/tissues.Organ-/tissue-specific expression was observed for FAD2c-1 (high expression in stem xylem), and FAD2d-1, FAD2f-1, FAD3c-1, and FAD3c-2 (high expression in roots).FAD2 plays an important role in the synthesis of PUFAs in nonphotosynthetic tissues, including roots [101,102].In flax roots, the FAD2d-1 and FAD2f-1 genes seemed to be especially important for FA synthesis.Thus, our study significantly improved the understanding of which FAD and SAD genes play key roles in fatty acid synthesis in various organs and tissues of flax.
We demonstrated that four SAD genes had the highest expression in embryos, endosperm, and capsules.SAD2-1 and SAD2-2 were also expressed in the majority of the analyzed organs/tissues but at a lower level than in capsules.The differences in SAD3-1 and SAD3-2 expression between various flax organs/tissues were more distinct, with extremely low/zero expression in certain samples.In most articles on flax, the authors analyzed the SAD2-1 and SAD2-2 genes [51,79,103,104] (according to the classification of You et al. [46]; called SAD1 and SAD2 in the earlier study [40]).However, SAD3-1 and SAD3-2 remained out of scope in their research.Nevertheless, poorly studied SAD3 could significantly contribute to the synthesis of oil FAs because of the high expression of these genes in flax seeds.Thus, the SAD3 genes deserve to be studied in detail.
FAD and SAD are known to be involved in the response to stressors [80,[105][106][107].We discovered the differential expression of FAD and SAD in flax plants in response to biotic and abiotic stress conditions.FAD2c-2, FAD2d-1, FAD3c-1, FAD3c-2, FAD3d-1, FAD3d-2, SAD3-1, and SAD3-2 changed expression levels on inoculation with the Fusarium species, while the FAD2d-1 and FAD3d-1 expression changed in response to drought.Although the difference between expression in susceptible and resistant flax genotypes was insignificant, these genes could be targeted in research on the stress response of flax plants.
Thus, the role of certain FAD2, FAD3, and SAD genes in the development of key flax plant characteristics could be elucidated from the analysis of their expression in various organs/tissues, at different developmental stages, and under different growth conditions.For instance, such research could establish the contribution of FAD2, FAD3, and SAD to the FA composition of flax oil.The information collected in the present study is the basis for developing effective flax genome editing procedures, as well as marker-assisted and genomic selection.These technologies are for the creation of flax varieties with a determined oil composition and require a solid theoretical background.In addition, recent research into different plant species aimed at FAD and SAD identification [10,[12][13][14]16,[109][110][111].Based on the analysis of gene sequences of several genotypes and further expression evaluation in a representative set of transcriptomic data, our approach can be effective in a broad range of studies on the FAD and SAD genes.
For the extracted sequences, phylogenetic analysis was carried out in MEGA X [113].Multiple alignment of the extracted sequences was performed using the MUSCLE algorithm with default parameters.Then, phylogenetic trees were constructed using the maximum likelihood method (default parameters, 1000 bootstrap replicates).

Figure 4 .
Figure 4. Expression of FAD2 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures4 and 5is common.Noted gene expression patterns of interest are highlighted in boxes.
Figure 4. Expression of FAD2 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures4 and 5is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 4 .
Figure 4. Expression of FAD2 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures4 and 5is common.Noted gene expression patterns of interest are highlighted in boxes.
Figure 4. Expression of FAD2 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures4 and 5is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 5 .
Figure 5. Expression of FAD2 genes in flax plants under different stress conditions.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures4 and 5is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 6 .Figure 6 .
Figure 6.Expression of FAD3 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Figure 6.Expression of FAD3 genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures6 and 7is common.Noted gene expression patterns of interest are highlighted in boxes.
is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 7 .
Figure 7. Expression of FAD3 genes in flax plants under different stress conditions.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures6 and 7is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 7 .
Figure 7. Expression of FAD3 genes in flax plants under different stress conditions.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures6 and 7is common.Noted gene expression patterns of interest are highlighted in boxes.

22 Figure 8 .
Figure 8. Expression of SAD genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.
Figure 8. Expression of SAD genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 8 .
Figure 8. Expression of SAD genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.
Figure 8. Expression of SAD genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 8 .
Figure 8. Expression of SAD genes in different flax organs/tissues.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 9 .
Figure 9. Expression of SAD genes in flax plants under different stress conditions.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.

Figure 9 .
Figure 9. Expression of SAD genes in flax plants under different stress conditions.(a) Common color scale for all genes.(b) Individual color scale for each gene.Blue (0.01 × Average)-White (Average)-Red (10 × Average) color scale is used.The expression data for organs/tissues and stress conditions were analyzed together (as in Supplementary TableS2), so the color scale in Figures8 and 9is common.Noted gene expression patterns of interest are highlighted in boxes.

Table 4 .
Coordinates of SAD genes in genomes of flax varieties YY5, 3896, Atlant, and CDC Bethune.

Table 4 .
Coordinates of SAD genes in genomes of flax varieties YY5, 3896, Atlant, and CDC Bethune.