Haplotype analysis and molecular marker development for the cold tolerance gene OsCTS11 at the seedling stage of rice

Low-temperature stress poses a significant challenge to the growth and yield of rice seedlings. Although quantitative trait loci (QTLs) have been mapped and underlying genes for cold tolerance identified, breeding efforts remain constrained by the lack of precise molecular markers. In this study, we analyzed 529 accessions from the 3K Rice Genomic Diversity Panel to investigate genetic variations in OsCTS11, a known negative regulator of cold tolerance in rice seedlings. Linkage disequilibrium (LD) analysis identified three critical LD blocks (BLOCK1-3) within OsCTS11, each containing four distinct haplotypes. Association analysis revealed that Hap4 in BLOCK1, Hap3 in BLOCK2, and Hap4 in BLOCK3 significantly increased seedling survival rates to 65.38%, 58.41%, and 51.48% respectively, predominantly in japonica subspecies. These beneficial haplotypes demonstrated adaptation to temperate zones (30°-40°N) and tropical highlands (800–1500 m elevation), consistent with the evolutionary progression of cold tolerance in japonica rice. The utility of KASP molecular markers based on SNP sites was validated through this study. Among 42 rice varieties screened, indica R676 and japonica Nangeng 5718, both possessing dominant haplotypes, exhibited higher survival rates compared to varieties lacking these haplotypes. Marker-assisted backcrossing facilitated the development of four novel cold-tolerant germplasms (YR05-YR08) incorporating advantageous OsCTS11 haplotypes. Notably, YR08 (Hap4 + Hap3 + Hap4) showed significantly improved seedling establishment under cold stress, illustrating the synergistic benefits of stacked haplotypes. This research underscores the potential of leveraging natural variation haplotypes to create precise molecular markers for identifying beneficial OsCTS11 haplotypes, providing a novel approach to exploiting negative regulatory genes in rice breeding programs.