Unraveling the Genetic Basis of Cold Tolerance in Peanut (Arachis hypogaea L.) via QTL Mapping and Identification of Ahcold18 as a Candidate RWP-RK Transcription Factor

Peanut (Arachis hypogaea L.) is a globally vital oil and cash crop. However, frequent cold stress severely compromises its yield stability. To address this challenge, we conducted quantitative trait locus (QTL) mapping for two cold tolerance-associated traits, namely relative emergence rate (RER) and relative emergence index (REI), across four distinct environments using a recombinant inbred line (RIL) population derived from the cold-tolerant landrace Silihong and cold-sensitive cultivar Jinonghei 3. Two core QTLs associated with cold tolerance were detected, including qRER6 stable across all environments with PVE of 4.91–5.15% and a co-localized QTL qRER18.1/qREI18.2 on chromosome 18 that governs both target traits with PVE of 14.56–14.71% and 10.34%. Through integrated analysis of QTL mapping and Weighted gene co-expression network analysis (WGCNA), Arahy.657RUG was identified as a candidate cold tolerance gene and designated Ahcold18. Differential expression analysis and heterologous overexpression in Arabidopsis thaliana under freezing stress (−9 °C) showed that Ahcold18 enhances plant survival under low-temperature stress, suggesting a general role in cold tolerance that warrants further investigation in the context of peanut chilling tolerance. A gene-based KASP marker (KASP-2374669), developed from variant sites within Ahcold18, showed preliminary association with RER and REI in the RIL population; however, further validation in diverse germplasm is required to confirm its utility for marker-assisted selection. This study provides a critical genetic resource and a precise technical tool for marker-assisted breeding of cultivated peanut with cold tolerance.