Rice adaptation to low temperatures involves changes in their genetic and physiological architecture, caused by complex and variable transcriptional regulatory networks to regulate between growth and stress resistance. Overwintering rice survives cold winter and germinates the following spring. The perennial and cold‐tolerant properties present great potential regarding rice breeding and food security. In this study, the cold‐tolerant overwintering cultivar rice, “glutinous rice 89‐1” (Gr 89‐1), and an indica cultivar rice, “R07,” were placed at 4°C at the tillering stage and sampled at 0, 12, 24, and 96 h, and recovery 7 days. Physiological indicators of each sample were measured, and transcriptome analysis was performed. The results revealed that the overwintering cultivar rice Gr 89‐1 exhibited a pattern of physiological parameters consistent with the cold‐tolerant phenotype, less membrane damage accumulation and water loss with increasing duration of low temperature, and a stable increase in peroxidase activities, catalase activities, and soluble sugar content. Transcriptome analysis of two cultivars at 0, 12, and 24 h under 4°C revealed different gene expression patterns to low temperatures. Compared with R07, gene ontology terms of nuclear lumen, endomembrane system, organelle membrane, and envelope were enriched in Gr 89‐1, suggesting an alteration of membrane composition during cold acclimation. Specific upregulated transcription factors and cold‐tolerance regulators indicated a coordinated response between signal transduction, energy regulation, cell cycle, and circadian rhythm, which might help maintain cell homeostasis and improve Gr 89‐1 survival under cold stress. Two candidate genes OsETR3 and NAC78 were screened out, they significantly upregulated at 12 and 24 h of cold treatment in Gr 89‐1 and were higher than R07, and they were involved in cell wall remodeling and endoplasmic reticulum homeostasis under stress, respectively, which may relate to the cold‐tolerant trait of Gr 89‐1.

In this study, a Glutinous rice 89-1 (Gr 89-1) × Shuhui 527 recombinant inbred line population (RIL) comprising 309 F₉-generations was used to screen gradient molecular markers. The phenotypic variation and distribution of eight agronomic traits obtained from multiyear and multilocation samples, as well as the network expression relationships between agronomic traits and molecular markers, were investigated. The results showed that there were 14 phenotypic lines with significant differences in the RILs, and the molecular testing results of most of the lines were consistent with the phenotype. The correlation degree between the first-level molecular markers and the eight agronomic traits was 100%. Excluding the correlations of third-level markers with grain width and grain length, the degree of correlation between molecular markers and agronomic traits decreased with an increase in marker levels. The RILs were divided into eight core populations and one approximate population, revealing genetic correspondence between agronomic traits and molecular markers.