Investigating cold tolerance mechanisms in rice seedlings: Alternative splicing, promoter analysis, and their applications for marker development

Cold stress harms rice seedlings, causing yield reduction. Enhancing cold tolerance at the seedling stage is crucial for rice breeding. OsFH10, OsFER1, ONAC045, and OsProT were selected to study gene expression including alternative splicing and promoter sequence analysis in rice seedlings. Under cold stress, the four genes exhibited alternative splicing showing intron retention isoforms, and displayed a consistent pattern of upregulation in a group of cold-tolerant varieties, while no isoforms were presented in a group of sensitive varieties. These intron retention isoforms might play a role in facilitating improved cold adaptation in rice seedlings. Promoter sequence analysis revealed polymorphisms of Single nucleotide polymorphisms (SNPs) or Insertions and Deletions (InDels) in ONAC045 and OsProT that distinguished cold-tolerant from cold-sensitive varieties. ONAC045 SNP and OsProT InDel markers were developed and validated using 159 rice lines. These markers were associated with cold tolerance (P < 0.05) and practical to use with general agarose gel. The ONAC045 marker showed high efficacy in predicting tolerance in rice germplasm seedlings, achieving an accuracy of 43.8%. Furthermore, the OsProT marker exhibited greater sensitivity towards cold-tolerant indica seedlings. Both markers have a wide distribution in all rice subspecies. Our findings elucidate the cold response mechanisms and provide insights into potential application in developing cold-tolerant indica rice varieties.