Expression of FAD and SAD Genes in Developing Seeds of Flax Varieties under Different Growth Conditions

1. Introduction

Flax (Linum usitatissimum L.) seed is one of the richest plant sources of omega-3 fatty acids, which are essential for humans and prevent the onset and progression of many diseases [1,2,3,4]. Flax varieties differ significantly in the fatty acid (FA) composition of the oil, especially in the content of linolenic acid (omega-3) [5,6,7]. Traditional flax varieties are high in linolenic acid (LIN, 50-60%), and are used in the pharmaceutical and paint industries [6,8]. Varieties with low (about 5%) and medium (30-40%) LIN content have been also developed and are promising for use in the food industry due to the greater resistance of oil to rancidity [6,8,9]. Linoleic acid (LIO, omega-6) and oleic acid (OLE, omega-9) are also important unsaturated fatty acids in flax seeds. There is an inverse relationship between LIO and LIN levels: high-LIN varieties have low LIO content and low-LIN varieties have high LIO content [7,10]. Differences in the OLE content between varieties are less pronounced and can be about 2-fold [7,10]. Varieties with higher OLE content are promising for the production of oxidation-stable edible oil, chemical feedstock, and biodiesel [8,9].

Desaturases are known to play a key role in the synthesis of flax fatty acids by introducing double bonds into the hydrocarbon chain. SAD (stearoyl-ACP desaturases) catalyze the conversion of stearic acid to oleic acid, FAD2 (fatty acid desaturases 2) catalyze the conversion of oleic acid to linoleic acid, and FAD3 (fatty acid desaturases 3) catalyze the conversion of linoleic acid to linolenic acid [11,12,13,14,15,16,17]. In flax, 25 genes of the SAD and FAD families were identified: SAD2-1, SAD2-2, SAD3-1, SAD3-2, FAD2a-1, FAD2a-2, FAD2b-1, FAD2b-2, FAD2c-1, FAD2c-2, FAD2d-1, FAD2d-2, FAD2e-1, FAD2e-2, FAD2f-1, FAD2f-2, FAD2g-1, FAD2g-2, FAD2h, FAD3a, FAD3b, FAD3c-1, FAD3c-2, FAD3d-1, and FAD3d-2 [18,19]. Mutations in the FAD3a and FAD3b genes are known to result in reduced linolenic acid content in oil [7,16,17,20,21,22,23,24]. The association of FAD3a and FAD3b expression with LIN and LIO accumulation was reported [16,17,25]. However, no correlation was found between the expression of SAD1, SAD2, FAD2a, FAD2b, FAD3a, and FAD3b and the FA composition of linseed oil in genotypes with the same desaturase isoforms but different FA composition [26]. Several studies investigated changes in expression levels of FAD and SAD genes during flax seed development and identified the stages at which these genes are most active: 20 days after flowering (DAF) for FAD3 family genes [17]; 16, 22, and 30 DAF for genes of SAD, FAD2, and FAD3 families with variation among genotypes [25]; 20 and 24 DAF for genes of SAD, FAD2, and FAD3 families [26]; 15 and 20 DAF for genes of FAD2 family [8]. The influence of growing conditions on linseed oil content and its FA composition is known, and the influence of temperature and humidity was particularly noted [10,27,28,29,30]. The above works contributed significantly to the understanding of the effects of genotype and environment on the expression of key FA synthesis genes during flax seed development. However, a holistic picture of how growing conditions affect the expression of FAD and SAD genes during seed development in different flax genotypes is lacking. It is also necessary to understand which genes from the SAD, FAD2, and FAD3 families have the highest expression levels in seeds and, therefore, could contribute most to linseed oil FA synthesis at specific stages of seed development in different genotypes. These questions were the focus of our work.

2. Results

2.1. Results of Transcriptome Sequencing of Flax Seeds

Sequencing of 266 flax seed transcriptome libraries was performed. Ten flax varieties, which differed in the FA composition of oil and the presence of mutations in the FAD3 genes leading to reduced LIN content, were used in the work (Table 1): high-LIN AGT 427, Atalante, Entre-Rios, Norlin, and Pechersky kryazh; mid-LIN Raciol and AGT 422; low-LIN AGT 981, AGT 1535, and Lola. Seeds were collected at the 3, 7, 14, 21, and 28 day after flowering (DAF) from plants grown under conditions of 16 °C and overwatering (hereafter referred to as 16 °C), 20 °C and optimal watering (hereafter referred to as 20 °C), 24 °C and underwatering (hereafter referred as 24 °C). No data were obtained for variety AGT 422 at the 21 and 28 DAF at 24 °C and variety Lola at the 28 DAF at 24 °C, which is because of the negative impact of elevated temperature and insufficient watering on these genotypes. Sequencing was performed in two biological replicates for samples collected at the 3, 7, 14, and 21 DAF, and in one biological replicate for samples collected at the 28 DAF. On average, 2 million paired-end reads were obtained for each transcriptome library.

To visualize the relatedness of the studied samples, an MDS plot was constructed based on the expression of all identified transcripts (Figure 1). The samples were grouped mainly by developmental stage (i.e., DAF) and growth conditions (16 °C, 20 °C, and 24 °C) rather than belonging to a specific genotype.

2.2. Expression of the FAD and SAD Genes in Flax Seeds

Expression of genes of FAD and SAD families, which are known to play an important role in linseed FA synthesis [14,16,18,19,21,24,31,32], was evaluated. From 25 flax genes of SAD and FAD families (SAD2-1, SAD2-2, SAD3-1, SAD3-2, FAD2a-1, FAD2a-2, FAD2b-1, FAD2b-2, FAD2c-1, FAD2c-2, FAD2d-1, FAD2d-2, FAD2e-1, FAD2e-2, FAD2f-1, FAD2f-2, FAD2g-1, FAD2g-2, FAD2h, FAD3a, FAD3b, FAD3c-1, FAD3c-2, FAD3d-1, and FAD3d-2) [18,19,24], the highest expression levels in seeds were for FAD3b, FAD3a, FAD2b-2, SAD3-1, SAD2-1, SAD2-2, SAD3-2, FAD2a-1, and FAD2a-2 (Supplementary Table S1). It can be assumed that these genes play the most important role in linseed oil synthesis among the FAD and SAD genes. For these genes, a more detailed analysis of expression profiles in different flax varieties grown at 16 °C, 20 °C, and 24 °C was performed.

2.3. Expression Profiles of the FAD3a and FAD3b Genes during Flax Seed Development under Different Temperature and Watering Conditions

FAD3 genes are known to play a key role in the conversion of LIO to LIN in linseed [16,17]. Among the FAD3 genes, expression of FAD3a and FAD3b was the highest in the studied samples, ten times higher than that of FAD3c-1, FAD3c-2, FAD3d-1, and FAD3d-2. In different flax varieties, the expression of FAD3a and FAD3b generally changed similarly during seed development under the same growth conditions (Figure 2 and Figure 3 respectively). Expression was minimal at the 3 DAF. The maximum expression of FAD3a and FAD3b genes at 16 °C was observed at the 21 or 28 DAF in most varieties. At 24 °C, the maximum expression of FAD3a and FAD3b genes was reached at the 14 DAF, after which a decrease was observed. At 20 °C, the maximum expression levels were observed at the 14 or 21 DAF, after which it decreased. It is worth noting that the maximum expression levels of FAD3a and FAD3b genes were similar at 16 °C, 20 °C, and 24 °C. Thus, increased temperature resulted in a shift of the maximum level of gene expression to earlier stages of seed development, while decreased temperature resulted in a shift to later stages of seed development.

Expression profiles of the FAD3a and FAD3b genes were very similar for the same variety under the same conditions (16 °C, 20 °C, and 24 °C). However, in varieties AGT 981, AGT 1535, and Lola, the level of FAD3a transcript was significantly lower than in the other varieties, while the level of FAD3b transcript was similar to the other varieties. Varieties AGT 981, AGT 1535, and Lola are known to have mutations in the FAD3a gene resulting in the stop codons and reduced LIN content [7]. Thus, the FAD3a transcript level was reduced in varieties with nonsense mutations of this gene. However, the situation was different for the FAD3b gene. Varieties AGT 422, AGT 981, AGT 1535, and Raciol, which carry the FAD3b mutation leading to the histidine-to-tyrosine substitution and LIN content reduction [7], had approximately the same expression level of this gene as the varieties without the mutation. It is worth noting that the expression level of FAD3b was slightly higher than that of FAD3a in all the varieties studied.

2.4. Expression Profiles of the FAD2b-2, FAD2a-1, and FAD2a-2 Genes during Flax Seed Development under Different Temperature and Watering Conditions

FAD2 genes play an important role in the synthesis of linoleic acid from oleic acid [14,15]. Among the FAD2 genes, the highest expression levels in flax seeds were found for FAD2b-2, FAD2a-1, and FAD2a-2 (Supplementary Table S1). The expression level of FAD2b-2 was several times higher than that of FAD2a-1 and FAD2a-2 in all varieties studied. The expression level of FAD2a-1 was slightly higher than that of FAD2a-2. It should be noted that the FAD2a-1 and FAD2a-2 genes have very similar sequences, which differ by only 7 SNPs, which could introduce some bias in the estimation of their individual expression patterns [19]. For FAD2b-2 (Figure 4), FAD2a-1 (Supplementary Figure S1), and FAD2a-2 (Supplementary Figure S2), as for FAD3a and FAD3b, cultivation at 16 °C resulted in altered expression profiles compared to 20 °C and 24 °C and a shift of the maximum expression levels to later stages of seed development (the 21 or 28 DAF). Expression profiles of FAD2a-1 and FAD2a-2 were very similar for the same variety under the same conditions, but slightly different from those of FAD2b-2.

All varieties showed drastic changes in expression levels of FAD2b-2, FAD2a-1, and FAD2a-2 during seed development. At the 3 DAF, FAD2b-2, FAD2a-1, and FAD2a-2 were barely expressed, but at the 14 DAF (at 20 °C and 24 °C) or later (at 16 °C), a dramatic increase in expression levels was observed. At the 28 DAF, there was a strong decrease in expression levels of the genes for all varieties at 20 °C and 24 °C, and there were differences in expression changes (increase, decrease, or retention compared to the 21 DAF) among varieties at 16 °C. A decrease in the maximum expression level of FAD2b-2 at 16 °C compared to 20 °C and/or 24 °C can also be observed for some varieties. Pechersky kryazh had the highest OLE content (31.8%) among the 10 examined flax varieties, which was 1.5-2.0 times higher than that in other varieties. In this variety, the expression level of FAD2b-2 was slightly lower than that in the majority of varieties studied. Expression level of FAD2b-2 was also slightly lower in Norlin, which had increased OLE content (22.2%), and Lola, which had decreased OLE content (12.9%). For FAD2a-1 and FAD2a-2, the differences in expression levels between Pechersky kryazh and other varieties were not very noticeable. At the same time, expression profiles of FAD2b-2, FAD2a-1, and FAD2a-2 in the variety Pechersky kryazh were slightly different from other varieties, and the maximum expression levels were shifted to earlier stages of seed development (this was clearly visible for FAD2a-1 and FAD2a-2 at 20 °C and for FAD2b-2 at 24 °C). Thus, the high OLE content in the variety Pechersky kryazh is unlikely related to the maximum FAD2 expression levels. However, the shift of the maximum expression levels of FAD2 genes to earlier stages of seed development could play a role in increasing the OLE content in linseed oil.

2.5. Expression Levels of the SAD Genes during Flax Seed Development under Different Temperature and Watering Conditions

SAD genes are involved in the conversion of stearic acid to oleic acid in flax seeds [11,12,13]. In flax, all four identified SAD genes (SAD2-1, SAD2-2, SAD3-1, and SAD3-2) were rather highly expressed (Supplementary Table S1), with the highest level for SAD3-1. Expression profiles of SAD2-1 (Figure 5) and SAD2-2 (Supplementary Figure S3) for the same variety under the same conditions were very similar. The same was true for SAD3-1 (Figure 6) and SAD3-2 (Supplementary Figure S4). However, some differences in expression profiles were observed between the SAD2-1/SAD2-2 and SAD3-1/SAD3-2 pairs for the same samples, although the overall trend in expression changes was similar. During the seed development, an increase in expression levels of SAD genes was initially observed under all studied conditions. Then, at 20 °C and 24 °C, a decrease in expression levels was observed at the 21 or 28 DAF, while at 16 °C, different varieties showed an increase, decrease, or retention of expression levels. At the same time, at 20 °C and 24 °C for SAD3-1 and SAD3-2, the maximum expression levels were reached at earlier developmental stages (the 14 DAF for most varieties) compared to SAD2-1 and SAD2-2 (the 21 DAF at 20 °C and a shift from the 14 to the 21 DAF at 24 °C for most varieties).

It can be noted that for SAD2-1/SAD2-2 some expression was observed even at the 3 DAF, in contrast to the SAD3-1/SAD3-2 and FAD genes, for which expression was practically absent at this stage. It is also interesting that expression changes during seed development (most notably at 20 °C) were more similar between SAD3-1/SAD3-2 and FAD2b-2 (reaching maximum levels at earlier stages) and between SAD2-1/SAD2-2 and FAD2a-1/FAD2a-2, FAD3a, and FAD3b (reaching maximum levels at later stages). In Pechersky kryazh with high OLE content in the oil, characteristics similar to those described above for the FAD2 genes were observed for the SAD genes. Thus, expression levels of SAD genes in the high-OLE variety Pechersky kryazh were close to those in the low-OLE variety Lola. However, in Pechersky kryazh, the SAD expression profiles were slightly different from other varieties, and the maximum expression levels were shifted to earlier stages of seed development (this was particularly noticeable for SAD2-1 and SAD2-2 at 20 °C and for SAD3-1 and SAD3-2 at 24 °C). Thus, the high OLE content in the variety Pechersky kryazh could be related not to its differences from other varieties in the maximum expression levels of SAD and FAD2 genes, but to the shift of the maximum expression levels of these genes to earlier stages of seed development.

3. Discussion

It is known that the key role in the synthesis of unsaturated fatty acids in linseed oil is played by genes of SAD, FAD2, and FAD3 families [11,12,13,14,15,16,17]. We analyzed the expression of these genes during seed development in a representative set of 10 flax varieties with different oil FA compositions grown under three temperature and watering conditions. In contrast to most previous works evaluating SAD and FAD gene expression in flax [8,16,17,18,25,26], we obtained individual expression data for all 25 known SAD and FAD genes, rather than expression levels just for a few genes from each family or common expression patterns for pairs of homologous genes. Due to the large number of works devoted to transcriptome analysis of different flax organs and tissues [33,34,35,36,37,38,39,40,41,42,43,44], we were previously able to determine which of the 25 SAD and FAD genes are expressed at high levels in flax seeds, and to estimate expression levels of these genes in other organs and tissues [19]. However, in that work, due to the lack of transcriptomic data, we were unable to assess the dynamics of expression of these genes during seed development, which was done in the present work for a representative set of flax varieties grown under different conditions affecting the FA composition of linseed oil.

The highest expression levels were detected for FAD3b, FAD3a, FAD2b-2, SAD3-1, SAD2-1, SAD2-2, SAD3-2, FAD2a-1, and FAD2a-2. In addition, expression levels of these genes changed tens and hundreds of times during seed development. In the early stages of development, expression of these genes was minimal, then a drastic increase was observed, but the dynamics of growth depended significantly on the growing conditions. The strongest increase was observed at 20 °C and 24 °C, with a maximum reached at the 14 or 21 DAF in most varieties, followed by a drastic decrease. At 24 °C compared to 20 °C, a shift of the maximum expression levels to earlier developmental stages was observed for the majority of highly expressed FAD and SAD genes. At 16 °C, the maximum expression levels of the analyzed genes were observed at later stages of seed development (predominantly at the 21 and 28 DAF), and the differences between varieties were quite pronounced. It is likely that among the FAD and SAD families, it is FAD3b, FAD3a, FAD2b-2, SAD3-1, SAD2-1, SAD2-2, SAD3-2, FAD2a-1, and FAD2a-2 genes with high maximum expression levels and drastic expression changes during seed development that play a key role in the FA synthesis of linseed oil.

It is also possible to hypothesize how variations in FAD and SAD expression are reflected in the composition of linseed oil when plants are grown under different temperature and humidity conditions. Wet and cold summers are known to increase LIN+LIO level in linseed oil, whereas hot and dry summers are known to increase OLE content [10]. In our study, the decrease in FAD2a-1 and FAD2a-2 expression at higher temperature (24 °C) occurred at earlier stages of seed development compared to 20 °C and especially 16 °C. This trend was less clear for FAD2b-2, but it was also present in some genotypes. Probably under higher temperatures and insufficient watering, the FAD2 genes do not have enough time to desaturate oleic acid to linoleic acid to the same extent as at lower temperatures, resulting in increased OLE and decreased LIO+LIN content. FAD genes are known to be involved in the response to a variety of stresses, including high and low temperatures [45]. The effects of increased and/or decreased temperatures on the expression of FAD genes were reported for banana [46], maize [47], Gossypium [48], cucumber [49], and some genotypes of soybean [50]. The effect of temperature on the production of LIN and LIO in olive was also shown [51]. For flax, we observed that temperature affects expression profiles of FAD and SAD genes, which could be reflected in the oil FA composition.

We found no correlation between expression levels of FAD2 or SAD genes and OLE content. Thus, among the genotypes we studied, there was a high-oleic variety Pechersky kryazh, and expression levels of FAD2 and SAD genes in it were close to those of the low-oleic variety Lola. At the same time, a shift of the maximum expression levels of FAD and SAD genes to earlier dates was observed in Pechersky kryazh compared to other varieties studied. This peculiarity could lead to a higher content of OLE in seeds of this variety. The strongest expression of FAD and SAD genes for this variety may occur during hotter summer periods than that for other varieties, which could further enhance the effect of increasing OLE content and decreasing LIO content. We grew flax plants under controlled conditions at constant temperatures, so differences in flowering time did not introduce a bias in the evaluation of expression levels. However, when plants are grown under field conditions, the contribution of differences in flowering time between genotypes could also be reflected in the oil FA composition, because seed maturation may occur at different temperature/watering conditions.

Among the SAD genes, the highest expression level was found for SAD3-1. The expression level of its homolog SAD3-2 was on average two times lower. Expression levels of homologous genes SAD2-1 and SAD2-2 were quite high, no more than 30% lower than that of SAD3-1. The expression level of SAD2-1 was slightly higher than that of SAD2-2. It can be assumed that the SAD3-1 gene plays the greatest role in the conversion of stearic acid to oleic acid in flax seeds, but SAD2-1, SAD2-2, and SAD3-2 also contribute significantly to this process.

Among the FAD2 genes, FAD2b-2 had the highest expression level in most samples. However, the expression level of its homolog FAD2b-1 was dozens of times lower. For the homologous FAD2a-1 and FAD2a-2, expression levels were quite similar, with a slightly higher level of FAD2a-1. FAD2b-2 is probably the key gene in the desaturation of oleic acid to linoleic acid in flax seeds, but FAD2a-1 and FAD2a-2 also contribute to this process. The role of FAD2b-1 in linoleic acid synthesis in linseed oil is probably insignificant, which is also characteristic of the other FAD2 genes studied, namely FAD2c-1, FAD2c-2, FAD2d-1, FAD2d-2, FAD2e-1, FAD2e-2, FAD2f-1, FAD2f-2, FAD2g-1, FAD2g-2, FAD2h.

Among the FAD3 genes, FAD3a and FAD3b genes were characterized by the highest expression levels. Expression levels of these genes were similar, but in varieties AGT 981, AGT 1535, and Lola with nonsense mutations in the FAD3a gene leading to reduced LIN content, the level of FAD3a transcript was ten times lower than the level of FAD3b transcript. The low level of FAD3a transcript in the flax genotype with mutation of this gene leading to stop codon was previously reported and it was explained by nonsense-mediated mRNA decay [17]. No decrease in the FAD3b transcript level was detected in varieties with FAD3b missense mutation leading to the LIN content reduction. The fact that a missense mutation of the FAD3b gene does not lead to a change in its expression compared to other varieties was also reported earlier [17]. It was also shown previously that several FAD3a and FAD3b gene isoforms carrying mutations encode non-functional enzymes that are unable to convert LIO to LIN [24]. The contribution of FAD3a and FAD3b to the formation of LIN from LIO is probably close, but the expression level of FAD3b was slightly higher than that of FAD3a, so the role of FAD3b in the synthesis of linolenic acid could be slightly higher. This assumption is supported by the higher LIN content in flax varieties with mutations in the FAD3a gene compared to varieties with mutations in the FAD3b gene [7].

We observed similar expression profiles for SAD3-1, SAD3-2, and FAD2b-2. Expression profiles of SAD2-1, SAD2-2, FAD2a-1, FAD2a-2, FAD3a, and FAD3b were also similar. For the first group of genes, maximum expression levels were reached at earlier stages of seed development compared to the second group. There could be common regulatory mechanisms for the genes in each group, and these groups of genes could contribute somewhat differently to the synthesis of linseed oil at different stages of seed development.

Thus, we were able to identify FAD and SAD genes with the highest expression in flax seeds, which probably play an important role in the synthesis of OLE, LIO, and LIN of linseed oil. Expression profiles of the highly expressed FAD and SAD genes we examined were quite similar during seed development under the same conditions. All FAD and SAD genes showed dramatic changes in expression levels during seed development. The dependence of expression profiles on growth conditions was also observed: lower temperatures shifted the maximum expression levels to later stages of seed development, while higher temperatures often resulted in shifts of maximum expression levels to earlier stages or an earlier decrease of expression during seed development. These changes could be reflected in the FA composition of linseed oil. In addition, genotype-dependent features of FAD and SAD expression were found. A correlation between the transcript level and LIN content was found for the FAD3a gene, but for other FAD and SAD genes investigated, no pronounced associations between expression levels and OLE, LIO, or LIN content were found. Our work is important for understanding the role of FAD and SAD genes in linseed fatty acid synthesis and the contribution of genotype and environment to the expression of these genes. From an applied point of view, the results obtained can be used in marker-assisted selection and genome editing to create flax varieties with the desired fatty acid composition in seed, as well as in the development of recommendations for the optimal conditions of oilseed flax cultivation to obtain oil with the specific FA composition.

4. Materials and Methods

4.1. Flax Varieties

We used plants of 10 flax varieties: AGT 422, AGT 427, AGT 981, AGT 1535, Atalante, Entre-Rios, Lola, Norlin, Pechersky kryazh (namely, l. 1-2 from k-2889 according to the catalog of the Federal Research Center for Bast Fiber Crops, selection from Pechersky kryazh, originator N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR)), and Raciol. AGT 981, AGT 1535, and Lola had low LIN content; Raciol and AGT 422 had medium LIN content; AGT 427, Entre-Rios, Norlin, Atalante, and Pechersky kryazh had high LIN content (Table 1). Low and medium LIN levels were associated with mutations in the FAD3a and FAD3b genes [7], data are shown in Table 1.

4.2. Growth Conditions

Flax plants were grown in 15-liter pots with soil pretreated with fungicide. pH was maintained at about 5.5, the optimum level for flax. 32 seeds were sown per pot and three pots were planted for each variety. All plants were kept in the same conditions for one month after planting. The plants were then transferred to three climate chambers with different temperature regimes for growth. In the first chamber, the temperature was maintained at 16 °C, and watering was done every day. In the second chamber, the temperature was 20 °C, and watering was done every two days. In the third chamber, the temperature was 24 °C, and watering was done every three days.

4.3. Collection of Plant Material and RNA Isolation

The flower was marked with the date on the day it opened (day of flowering) and the data were recorded in a table. Seeds were collected at the 3, 7, 14, 21, and 28 day after flowering (DAF). For each variety, at least 10 samples were collected for each temperature/watering condition and each developmental stage analyzed. Seeds collected from individual capsules were placed in tubes and immediately frozen in liquid nitrogen. Plant material was placed in a low-temperature freezer at -70 °C.

For RNA isolation, seeds were first ground to a fine powder using disposable homogenization pestles (Helicon, Russia) inserted into a DeWalt DCD701D2 cordless drill (DeWalt, Germany) at a speed of 1200-1500 rpm in 1.5 mL tubes in liquid nitrogen without thawing the sample. RNA isolation was performed according to the protocol of Wang et al. [52] with some modifications. Briefly, 1 mL of pre-warmed to 65 °C CTAB lysis buffer (2% CTAB (neoFroxx GmbH, Germany), 2% PVP K30 (Pan-Reac AppliChem, Germany), 100mM Tris HCl pH 8.0 (Thermo Fisher Scientific, USA), 25mM EDTA (Thermo Fisher Scientific), 2 M NaCl (Scharlab, Spain), 2% β-mercaptoethanol (BioRad, USA)) was added to the homogenized sample. The homogenate was incubated in a TDB-120 thermostat (Biosan, Latvia) at 65 °C for 30 min with mixing every 10 min. Then, five identical samples (the same variety, growing condition, and developmental stage) were mixed as follows: 350 µl of homogenate was taken from each sample using a 1000 ml wide-bore pipette tip and added to a 2 ml tube. Next, 500 μl of homogenate was taken from the pool (the rest was frozen in liquid nitrogen) and 500 μl of CTAB lysis buffer pre-warmed to 65 °C was added. In the next step, an equal volume of cold chloroform (Acros Organics, USA) was added to the homogenate, vortexed for 30 sec, and then centrifuged on a microcentrifuge 5418R (Eppendorf, Germany) for 20 min at 10000 g and 4 °C. The aqueous phase was transferred to clean tubes, then an equal volume of cold chloroform was added, vortexed for 30 sec, and centrifuged for 10 min at 10000 g and 4 °C. The aqueous phase was transferred to clean tubes and 1/2 volume of 96% ethanol was added and mixed until homogeneous. Total RNA was then purified using the CleanRNA Standard Kit (Evrogen, Russia) according to the manufacturer's protocol with a DNAase I treatment step from the RNase-Free DNase Set (Qiagen, USA). RNA quality and concentration were evaluated using a gel electrophoresis, 2100 Bioanalyzer (Agilent Technologies, USA), and Qubit 4 fluorometer (Thermo Fisher Scientific). Only non-degraded RNA samples with a concentration of at least 20 ng/μL were used for further work.

4.4. Preparation and Sequencing of cDNA Libraries

Preparation of cDNA libraries for sequencing of flax seed transcriptomes was performed using the QIAseq Stranded mRNA Select Kit (Qiagen) according to the manufacturer's protocol. RNA from seeds of 10 flax varieties (AGT 422, AGT 427, AGT 981, AGT 1535, Atalante, Entre-Rios, Lola, Norlin, Pechersky kryazh, and Raciol) harvested at the 3, 7, 14, 21, and 28 DAF from plants grown under three different temperature/watering conditions were used. For the 3, 7, 14, and 21 DAF, cDNA libraries were prepared in 2 replicates, and for the 28 DAF – in 1 replicate (because of the difficulty of obtaining RNA of high quality and sufficient quantity for seeds of late developmental stages). The quality of the obtained cDNA libraries (agreement of the length of the obtained libraries with the expected ones and absence of adapter dimers) was evaluated on a 2100 Bioanalyzer (Agilent Technologies), and the concentration was evaluated on a Qubit 4 fluorometer (Thermo Fisher Scientific).

The resulting cDNA libraries (266 libraries in total, as the libraries for the variety AGT 422 at the 21 and 28 DAF at 24 °C and the variety Lola at the 28 DAF at 24 °C were excluded from the analysis due to low quality) were mixed equimolarly and sequenced on a NextSeq 2000 instrument (Illumina, USA) using the NextSeq 2000 P3 Reagents (100 Cycles) kit (Illumina) in 51 + 51 nucleotide format.

4.5. Expression Analysis

Raw reads were processed using Trimmomatic 0.38 [53]. The data was checked for the presence of adapter sequences, trimmed by quality (TRAILING:24 SLIDINGWINDOW:4:14), and filtered by length (MINLEN:40). Then, the PPline tool was used for expression analysis [54]. Transcriptomic reads were aligned to the variety Atlant genome (GCA_014858635.1 in NCBI Genome) [55], and counts per million (CPM) values were calculated for 25 genes: SAD2-1 (Atlant transcript H1233_031351), SAD2-2 (H1233_038408), SAD3-1 (H1233_054424), SAD3-2 (H1233_039970), FAD2a-1 (H1233_058938), FAD2a-2 (H1233_061927), FAD2b-1 (H1233_075799), FAD2b-2 (H1233_078572), FAD2c-1 (H1233_075801), FAD2c-2 (H1233_078571), FAD2d-1 (H1233_041596), FAD2d-2 (H1233_078570), FAD2e-1 (H1233_075803), FAD2e-2 (H1233_078569), FAD2f-1 (H1233_075804), FAD2f-2 (H1233_078567), FAD2g-1 (H1233_075805), FAD2g-2 (H1233_078566), FAD2h (H1233_078565), FAD3a (H1233_027729), FAD3b (H1233_038272), FAD3c-1 (H1233_054813), FAD3c-2 (H1233_039845), FAD3d-1 (H1233_041596), and FAD3d-2 (H1233_041890).