Haplotype analysis and molecular marker development of the COLD1 for cold stress tolerance at the germination stage in rice.

Cold tolerance during the germination stage is a critical agronomic trait that influences the geographic adaptation and yield stability of rice. In this study, we systematically analyzed genetic variations of the COLD1 gene using the 3 K Rice Core Collection, revealing significant associations between haplotypes and cold tolerance at the germination stage. We identified two key SNPs (SNP145938700 and SNP145938916) in the upstream regulatory region of COLD1, which stratified the population into three distinct haplotypes: Hap1, Hap2, and Hap3. An association analysis of 493 germplasms subjected to cold stress demonstrated that Hap2 (SNP145938700-AA/SNP145938916-TT) exhibited superior cold tolerance, with a seedling survival rate of 62.76 %, significantly higher than that of Hap1 (55.88 %) and Hap3 (48.25 %) (P < 0.01). Haplotype distribution analysis across 2,840 germplasms revealed that Hap2, predominantly found in japonica subspecies (TEJ/TRJ), has a latitudinal adaptation advantage, suggesting its genomic architecture harbors unique cold-adaptive modules. We developed two kompetitive allele-specific PCR (KASP) markers, Cold1-1-kasp and Cold1-2-kasp, to differentiate between Hap1 (TT/GG), Hap2 (AA/TT), and Hap3 (AA/GG) with 100 % accuracy. Validation using 35 rice germplasms for production applications confirmed that carriers of Hap2 exhibited significantly higher seedling survival rates than those of Hap3. Furthermore, molecular marker-assisted selection (MAS) was employed to introgress Hap2 from the cold-tolerant donor Meixiangzhan 2 into the cold-sensitive cultivar R9311, resulting in the novel cold-tolerant germplasm RY218. In BC2F5 generations, RY218 achieved a seedling survival rate of 69.17 %, representing a seven-fold improvement over the recurrent parent. This study establishes COLD1 Hap2 as a key genetic determinant of germination stage cold tolerance and provides robust KASP markers for precision breeding in rice.