Seeds of linseed line 3896 were provided by the Institute for Flax (Torzhok, Russia). To obtain transcriptome data, we collected a set of organs ( Table 1 ; Figure 1 ) of line 3896 plants at different stages of vegetation under optimal growth conditions described in the next subsection.

Flax seeds were sterilized in 1% sodium hypochlorite and 96% ethanol, after which the seeds were washed twice with water and planted in 0.05% fungicide Maxim (Syngenta, Gaillon, France) solution in Petri dishes for 7 days. Seedlings were planted in the soil and continued to grow under greenhouse conditions at 20°C and ~50-70% relative humidity with regular watering.

For transcriptome analysis, a set of different organs/tissues at different development stages was collected. The plant parts and ontogenetic stages used in the study are listed in Table 1 . Each of the samples was collected in two biological replicates. Moreover, each sample was a pool of organs from 2-10 different plants (except capsules and seeds), which is necessary to level out differences between samples and be able to capture trends common to the species (Takele Assefa et al., 2020). The flowers were marked with the date of the day it opened (day of flowering). Seeds from the same capsules were pooled. Capsules were not pooled. The age of the plants at the time of each collection and the collection conditions are shown in Table 1 . Samples were collected in the middle of the day between 12 and 15 h under similar conditions to smooth the influence of circadian rhythms on gene expression profiles. Samples were collected in liquid nitrogen and stored at -70°C.

Samples #1-6 and #8-18 were grinded using a TissueLyser II homogenizer (Qiagen, Hilden, Germany) with the addition of 3 ceramic beads for two minutes. The harder samples (#7 and #19-28) were homogenized using a disposable pestle inserted in a DeWALT DCD701D2 cordless drill/driver (DeWALT, Towson, MD, USA) at 1200-1500 rpm in 1.5 ml tubes in liquid nitrogen to a fine powder, without allowing the sample to thaw. RNA isolation from samples #1-18 was performed using the Quick-RNA Miniprep Kit (Zymo Research, Irvine, CA, USA). RNA isolation from capsule and seed samples (#19-28) was performed by CTAB with modifications described previously (Pushkova et al., 2024). After that, total RNA was additionally cleaned using the CleanRNA Standard kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol with a DNAase I treatment step from the RNase-Free DNase Set (Qiagen). The quality of isolated RNA was checked by horizontal electrophoresis in 2% agarose gel and using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and concentrations were measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

The cDNA libraries were prepared using the QIAseq Stranded mRNA Select Kit (Qiagen) according to the manufacturer’s protocol. The quality of the obtained cDNA libraries (agreement of the length of the obtained libraries with the expected one and the absence of adapter dimers) was assessed on a Qsep1-Plus capillary electrophoresis system (BiOptic, New Taipei City, Taiwan), and the concentration was evaluated on a Qubit 4 fluorometer (Thermo Fisher Scientific). The cDNA libraries (all samples from Table 1 ) were mixed equimolarly and sequenced on a NextSeq 2000 instrument (Illumina, San Diego, CA, USA) using the NextSeq 2000 P3 Reagents (100 Cycles) kit (Illumina) in 51 + 51 nucleotide format.

RNA-Seq reads were trimmed with fastp 0.23.4 using default parameters (Chen et al., 2018). Structural annotation for the flax line 3896 genome assembly (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_030674075.2/, accessed on 12 October 2024) (Dvorianinova et al., 2023a) was performed using BRAKER3 3.0.8 (Gabriel et al., 2024). This process utilized our RNA-Seq transcriptome data in combination with known Viridiplantae sequences from OrthoDB protein database for gene prediction (Kuznetsov et al., 2023). Functional annotation of the predicted genes was carried out using a local version of InterProScan 5.69-101.0 to assign functional domains and predict gene functions (Jones et al., 2014). The completeness of the annotation was assessed using BUSCO 5.7.1 in protein mode (the eudicots_odb10 dataset). Default parameters of program settings were used in the data analysis.

Gene expression analysis was performed with PPline (Krasnov et al., 2015) with default parameters of program settings and included the following steps:

Custom Gene Ontology (GO) annotation for line 3896 was constructed using EggNOG-mapper (–tax_scope 33090) (emapper 2.1.12, eggNOG DB version: 5.0.2) for the longest proteins in the constructed gene annotation (Huerta-Cepas et al., 2019; Cantalapiedra et al., 2021). Differential gene expression analysis was conducted for a balanced subset of flax samples: mature leaf blades from the shoot at 10 cm from the top at 30 days after germination (DAG), stem fragments 1-3 cm from the top at 30 DAG, roots of seedlings at 5 DAG, hypocotyls of seedlings at 5 DAG, mature flower carpels before pollination at 56 DAG, capsule without seeds at 14 days after flowering (DAF), seeds without capsule at 14 DAF, shoot apical meristems (SAM) of seedlings at 5 DAG. Gene expression in each tissue was compared to that in the other tissues from the subset of flax samples. Genes with FDR (QLF) < 0.05 and logFC > 1.5 were selected for gene enrichment analysis. Gene enrichment was performed using the constructed annotation, the selected gene lists, and the topGO 2.54.0 package (Alexa and Rahnenfuhrer, 2023).

This article presents data of transcriptome analysis of 28 organs/tissues of flax line 3896: 7 samples of vegetative organs of actively growing individuals, 4 samples of seedlings, 7 samples of various parts of generative organs at flowering stage, and 10 samples of fruits and seeds at different stages of maturation ( Table 1 ; Figure 1 ). We set out to cover all growth stages of flax and all key events in the development of valuable flax traits. From 5.4 to 20.5 million raw reads (51 + 51 bp) were obtained for each sample on the Illumina platform (two biological replicates were sequenced for each sample). The raw data were deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1172129.

After trimming, the reads were mapped to the genome assembly of line 3896, and on average ~95% of the reads for each sample were mapped (on average ~90% were uniquely mapped), confirming the high quality of the transcriptome data.

We collected extensive data on gene expression in 28 organs/tissues of flax line 3896 at different development stages. Using the obtained transcriptome data and the Viridiplantae protein sequence database, we annotated the line 3896 genome assembly with BRAKER3: 39,055 genes and 45,154 transcripts were predicted, and 37,787 of these genes were annotated using InterProScan ( Supplementary Data Sheet S1 ). The high completeness of annotation was achieved according to BUSCO (Benchmarking Universal Single-Copy Orthologs) – 95.6% (eudicots). Notably, 22.2% of all BUSCO were complete and single-copy and 73.4% were complete and duplicated. Such a high percentage of duplicated BUSCO was expected for an ancient tetraploid (Bolsheva et al., 2017).

Utilizing the obtained annotation of line 3896, we performed an analysis of our transcriptome data, which resulted in the identification of genes exhibiting tissue-specific and development stage-specific expression patterns within flax organs/tissues. This analysis led to the generation of a comprehensive transcriptome map for line 3896. To present the data in a convenient format for further analysis, we used PPline and RTrans (https://github.com/gskrasnov/RTrans, accessed on 17 October 2024). It was applied to evaluate the expression levels of the identified genes as read counts per million reads (CPM). Our transcriptome map is summarized in Supplementary Table S1 and presented as a heatmap ( Supplementary Figure S1 ).

During the gene pathway enrichment analysis, the following was found out. For leaves collected at a distance of 10 cm from the apex, compared to the other organs/tissues, the GO terms were represented by the processes of photosynthesis, carbohydrate metabolism, plastid organization, electron transport chain, pigment synthesis, and transmembrane transport. These processes are characteristic of the main photosynthetic organ of the plant in the active phase (Müller and Munné-Bosch, 2021; Leister, 2023). In the stem fragment, located 1-3 cm from the top, the processes of vascular tissue histogenesis, vascular and phloem transport, stem morphogenesis, and response to auxin synthesized in the apical meristem predominated. This is logical, since we were dealing with an axial organ whose main function is the transport of metabolites, and the incision was made close to the site of differentiation (Yoshida et al., 2009; Kułak et al., 2023). It was determined that the major pathways in the seedling root included the processes of water and solute transport, root hair formation and growth, response to chemical and mechanical stimuli, and metabolism of auxin, other hormones, and secondary metabolites, which are the main processes occurring in the roots of vascular plants (Vissenberg et al., 2020; Li et al., 2021; Castillo-Jiménez et al., 2023). GO analysis of the hypocotyl transcriptome revealed differential expression of genes related to pathways of amino acid biosynthesis, as well as active regulation of biosynthetic processes, brassinosteroid metabolism, and cell growth. These findings indicate the presence of active development processes in the axial organs of seedlings (Favero et al., 2021). GO analysis for flax pistil demonstrated the representation of genes that are associated with the formation and development of generative structures, pollination, and pollen tube growth. Furthermore, it demonstrated representation of genes associated with active ion metabolism, which is necessary for directed pollen tube growth (Zhou et al., 2022). GO analysis for a capsule (14 DAF) indicated that the GO terms associated with the formation of secondary cell wall, synthesis of its components, and lignification prevailed. These processes enable the preparation of dry fruits for opening and seed dispersal (Seymour et al., 2013). Additionally, catabolism of organic compounds used for cell wall construction and seed maturation is active in ripening capsules. The differential expression pattern of flax seeds (14 DAF) was dominated by processes related to seed and fruit development and maturation, lipid storage and fatty acid synthesis, as well as abscisic acid (ABA) metabolism and regulation. Oil accumulation corresponds to the primary function of the seed as a reproductive organ, and ABA regulates its maturation (Sano and Marion-Poll, 2021; Dvorianinova et al., 2023b). The shoot apical meristem displayed a hallmark pattern indicative of actively dividing cells. The process of SAM is characterized by the macromolecule biosynthesis, ribosome assembly, translation, RNA processing, DNA reparation, organelle formation, nucleosome assembly, and chromatin remodeling (Xue et al., 2020; Burian, 2021). Thus, the results of the gene enrichment analysis of the subset of flax samples in general looked logical and confirmed the adequacy of the obtained data. The results are presented in detail in Supplementary Table S2 .