Rye (Secale cereale), a cereal crop with high cold tolerance, serves as an ideal model for investigating plant cold adaptation mechanisms. Despite recent progress in identifying numerous genes and metabolic changes associated with cold tolerance, the detailed regulatory networks and coordinated interactions between metabolic pathways under low-temperature stress in rye remain unclear. In this study, we focused on the winter rye variety “Winter” and systematically explored its metabolic regulatory responses to cold stress through a combination of low-temperature treatments, phenotypic observations, antioxidant enzyme activity assays, and transcriptomic analysis. Four rye varieties (“Winter”, HZHM3, HZHM8, and “Victory”) were compared for cold tolerance, with the results indicating that “Winter” and HZHM3 exhibit superior cold tolerance. Physiological analysis revealed that after 12 h of exposure to −4 °C, the activities of catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) in “Winter” were significantly upregulated, displaying an initial increase followed by a decline over time. Transcriptomic sequencing identified 1643 differentially expressed genes (DEGs), and GO, KEGG, and GSEA enrichment analyses highlighted the critical roles of carbohydrate metabolism (ko00630) and amino acid metabolism (ko00250) pathways in the cold stress response. These pathways are interconnected through key metabolic intermediates such as L-glutamate, collectively regulating cold adaptation. Furthermore, based on the transcriptomic data, we identified and developed molecular markers associated with cold tolerance, detecting 10,846 EST-SSR and 250,116 EST-SNP markers. We successfully developed 13 EST-SSR primer pairs applicable to rye and 7 KASP markers. Notably, the KASP-665 marker effectively distinguishes between winter and spring rye, providing a reliable tool for marker-assisted selection in cold tolerance breeding. This study not only elucidates the metabolic regulatory mechanisms of rye under low-temperature stress but also provides a solid theoretical and technical foundation for future cold-tolerance breeding programs.

Wheat is a crucial food crop, and low-temperature stress can severely disrupt its growth and development, ultimately leading to a substantial reduction in wheat yield. Understanding the cold-resistant genes of wheat and their action pathways is essential for revealing the cold-resistance mechanism of wheat, enhancing its yield and quality in low-temperature environments, and ensuring global food security. Rye (Secale cereale L.), on the other hand, has excellent cold resistance in comparison to some other crops. By studying the differential responses of different rye varieties to low-temperature stress at the transcriptome level, we aim to identify key genes and regulatory mechanisms related to cold tolerance. This knowledge can not only deepen our understanding of the molecular basis of rye’s cold resistance but also provide valuable insights for improving the cold tolerance of other crops through genetic breeding strategies. In this study, young leaves of two rye varieties, namely “winter” rye and “victory” rye, were used as experimental materials. Leaf samples of both types were treated at 4 °C for 0, 6, 24, and 72 h and then underwent RNA-sequencing. A total of 144,371 Unigenes were reconstituted. The Unigenes annotated in the NR, GO, KEGG, and KOG databases accounted for 79.39%, 55.98%, 59.90%, and 56.28%, respectively. A total of 3013 Unigenes were annotated as transcription factors (TFs), mainly belonging to the MYB family and the bHLH family. A total of 122,065 differentially expressed genes (DEGs) were identified and annotated in the GO pathways and KEGG pathways. For DEG analysis, 0 h 4 °C treated samples were controls. With strict criteria (p < 0.05, fold-change > 2 or 1), 122,065 DEGs were identified and annotated in GO and KEGG pathways. Among them, the “Chloroplast thylakoid membrane” and “Chloroplast” pathways were enriched in both the “winter” rye and “victory” rye groups treated with low temperatures, but the degrees of significance were different. Compared with “victory” rye, “winter” rye has more annotated pathways such as the “hydrogen catabolic process”. Although the presence of more pathways does not directly prove a more extensive cold-resistant mechanism, these pathways are likely associated with cold tolerance. Our subsequent analysis of gene expression patterns within these pathways, as well as their relationships with known cold-resistance-related genes, suggests that they play important roles in “winter” rye’s response to low-temperature stress. For example, genes in the “hydrogen catabolic process” pathway may be involved in regulating cellular redox balance, which is crucial for maintaining cell function under cold stress.