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