Integrating molecular markers and phenotypic analysis to assess cold tolerance in rice germplasm

Rice (Oryza sativa L.) is a crucial staple food for most of the world's population. However, it is highly vulnerable to low temperatures, which can induce growth retardation and yield loss. In this study, we aimed to develop SNP- and Indel-based molecular markers for the key cold tolerance-related genes HAN1, COLD11, and COLD1. The HAN1 marker was designed using a KASP assay, which was effective for fluorescence-based detection, whereas COLD11 and COLD1 markers were gel electrophoresis-compatible, enabling easy application without complex equipment. Considering the polygenic nature of cold tolerance, we analyzed combined markers, which exhibited enhanced prediction accuracy compared to single-marker analysis. Based on these markers, we categorized 372 rice cultivars into seven genotypic groups and assessed their genotypic and phenotypic data. The cold-tolerant HAN1 genotype was absent in the Tongil and indica cultivars but conferred the highest cold tolerance to japonica cultivars, highlighting the crucial role of HAN1 in the cold stress response. The COLD1 genotype and GCG repeat number of COLD11 are crucial for cold tolerance. Analysis of a doubled haploid population derived from a cross between the '93-11' and 'Milyang352' confirmed that the number of COLD11's GCG repeats significantly influence cold tolerance, followed by COLD1. Combining multiple cold-resistant alleles improved overall tolerance and post-stress recovery. Identifying additional alleles associated with cold stress resistance could aid in the selection of Tongil cultivars with enhanced cold tolerance. These markers could potentially contribute to breeding programs for the identification and selection of cold-tolerant rice varieties.