Transcriptome analysis of rice seedling under cold stress using RNA sequencing

Rice yields are threatened by climate change, particularly cold stress during the seedling stage. This study aimed to identify genes contributing to cold tolerance in rice seedlings. Using RNA sequencing, we identified cold-responsive genes in cold-tolerant and cold-sensitive varieties at the seedling stage after cold stress at 6-8 °C for 24 hours. This period provides different leaf rolling and withering of distinct seedling varieties. A total of 748 differentially expressed genes (DEGs) unique to B30 were identified, with a predominance of upregulated transcription factors (bZIP, bHLH, WRKY, NAC, and AP2/EREBP). 6 DEGs (OsFH10, ONAC045, SnRK2, OsISA1, OsFER1, and OsProT) exhibited distinct expression patterns in cold-tolerant varieties compared to cold-sensitive ones using RT-PCR. We further investigated their expression levels, methylation status, alternative splicing, and promoter sequences, in cold-stressed seedlings. Notably, all six genes displayed various transcript isoforms in cold-tolerant varieties under cold stress, due to intron retention. Promoter sequence analysis revealed single nucleotide polymorphisms (SNPs) or insertions/deletions (InDels) in SnRK2, ONAC045, and OsProT that differentiated cold-tolerant from cold-sensitive varieties. These polymorphisms could influence promoter activity and gene expression. Subsequently, ONAC045 SNP and OsProT InDel markers were developed and validated using 159 rice germplasm lines for cold tolerance prediction. The ONAC045 marker was extremely effective (43.8 %) in predicting cold tolerance but displayed lower sensitivity in cold-tolerant indica rice. Conversely, the OsProT marker offered slightly better sensitivity for indica rice. Both markers exhibited widespread distribution across rice subspecies. Our findings elucidate the cold response mechanisms of these genes and provide insights into their potential application in developing cold-tolerant rice varieties for future agricultural breeding.