Transcriptome analysis of flax (Linum usitatissimum L.) undergoing osmotic stress

Flax (Linum usitatissimum L.), an important industrial crop, is frequently decimated by pathogens, insect pests and adverse environmental conditions. Identification of flax resistance genes would facilitate development of varieties resistant to such diverse insults. Recently, a powerful method, high-throughput RNA sequencing (RNA-seq) technology, has been used effectively for transcript analysis and resistance gene discovery. In order to discover genes important for osmotic stress responses in flax, we performed transcriptome sequencing and screened differentially expressed unigenes (DEUs) of seedlings grown under normal and PEG 6000-induced stress treatments. Eight flax cDNA libraries were constructed and randomly sequenced using an Illumina technique and included one control sample and three treatment samples, each studied in duplicate. A total of 471,208,288 clean reads were obtained, of which 385,803,673 (81.88%) unique mapped reads and 23,042,410 multiple mapped reads were aligned to the Arabidopsis thaliana reference genome. Ultimately, 2533 out of 3922 unigenes were annotated using homology searches of KEGG Orthology (KO), PFAM, Gene Ontology (GO) and COG/KOG databases. Of these, 239 unigenes were differentially expressed (with at least 2-fold expression changes, FDR < 0.01) among three different PEG 6000-treatment durations compared with untreated control. Eight, five, four, three and one unigenes were assigned to NAC, LEA, WRKY, ERF and BLIP transcription factor families, respectively. For functional classification and pathway assignments, the 239 unigenes were mapped to three Gene Ontology (GO) categories including cellular component, molecular function and biological process, with 9, 10 and 19 functional terms for each category, respectively. In addition, these unigenes were assigned to 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, we validated the expression profiles of six DEUs using qRT-PCR to further confirm the accuracy of the RNA-seq data. To our knowledge, this study is the first systematic transcriptome analysis of flax osmotic stress response genes. The results reported here should aid identification of functional genes and putative pathways involved in flax osmotic resistance. Ultimately, this work should provide a reference map for discovery of additional stress tolerance genes useful for molecular breeding of flax varieties tolerant to a variety of stresses.