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Abstract

This study aims to elucidate the molecular mechanisms underlying cyanogenic glycoside accumulation in flax. As an important oil and fiber crop, the nutritional value of flax is compromised by the toxicity of cyanogenic glycoside. To clarify the key genetic regulators and temporal patterns of cyanogenic glycoside biosynthesis, transcriptomic sequencing was performed on seeds from high- and low-cyanogenic glycoside flax varieties (‘MONTANA16’ and ‘Xilibai’) at three developmental stages: bud stage, full flowering stage, and capsule-setting stage. A total of 127.25 Gb of high-quality data was obtained, with an alignment rate exceeding 87.80%. We identified 31,623 differentially expressed genes (DEGs), which exhibited distinct variety- and stage-specific expression patterns. Principal component analysis (PCA) and hierarchical clustering demonstrated strong reproducibility among biological replicates and revealed the seed pod formation stage as the period with the most significant varietal differences, suggesting it may represent a critical regulatory window for cyanogenic glycoside synthesis. GO and KEGG enrichment analyses indicated that DEGs were primarily involved in metabolic processes (including secondary metabolism and carbohydrate metabolism), oxidoreductase activity, and transmembrane transport functions. Of these, the cytochrome P450 pathway was most significantly enriched at the full bloom stage (H2 vs. L2). A total of 15 LuCYP450 and 13 LuUGT85 family genes were identified, and their expression patterns were closely associated with cyanogenic glycoside accumulation: In high-cyanogenic varieties, LuCYP450-8 was continuously upregulated, and LuUGT85-12 was significantly activated during later stages. Conversely, in low-cyanogenic varieties, high expression of LuCYP450-2/14 may inhibit synthesis. These findings systematically reveal the genetic basis and temporal dynamics of cyanogenic glycoside biosynthesis in flax and highlight the seed pod formation stage as a decisive regulatory window for cyanogenic glycoside synthesis. This study provides new insights into the coordinated regulation of cyanogenic pathways and establishes a molecular foundation for breeding flax varieties with low CNG content without compromising agronomic traits.