Discovery of cold tolerance genes and favorable alleles in Kam sweet rice across various growth stages.

key messageCombined association and selection sweep analyses identified OsCTD2 and OsLTPL159 as key candidate genes associated with cold tolerance at both germination and seedling stages. Cold sensitivity in rice leads to significant yield losses. Identifying cold-tolerant germplasm and uncovering cold tolerance genes are essential for developing resilient rice varieties. Kam Sweet Rice (KSR) is notable for strong cold tolerance, which is an invaluable genetic resource for identifying such genes. In this study, we phenotyped cold tolerance across various growth stages using 104 KSR accessions and 268 other rice landraces. Genome-wide association studies (GWAS) identified 89 loci significantly associated with cold tolerance: 57 loci at the germination stage, 9 at the bud stage, and 28 at the seedling stage, with 61 loci (69%) being newly discovered. Through association and selection sweep analyses, we identified two high-confidence candidate genes, OsCTD2 and OsLTPL159, associated with cold tolerance at both germination and seedling stages. Haplotype analysis revealed significant differences in cold tolerance grade and survival rate among various haplotypes of these genes, with superior haplotypes predominantly present in KSR. RNA-seq and qRT-PCR results showed that the superior haplotypes of OsCTD2 and OsLTPL159 exhibited significantly higher expression in cold-tolerant accessions under cold stress, whereas no significant differences were observed in cold-sensitive accessions with the inferior haplotypes. These results indicated that OsCTD2 and OsLTPL159 are involved in cold stress response in rice. Additionally, we identified two other promising candidate genes and their superior haplotypes for cold tolerance: OsGRS7 at the germination stage and OsBSR6 at the bud stage. Our findings provide a solid foundation for cloning cold tolerance genes and offer insights for designing molecular breeding strategies.