Peanut is a vital cash crop globally, and enhancing its seed protein content is essential for nutritional security. However, the genetic basis of seed protein content in peanut remains unclear. In this study, bulked segregant analysis combined with next-generation sequencing (BSA-Seq) was employed to isolate a candidate gene, AhSPC, associated with peanut seed protein content. Its function was then characterized in Arabidopsis thaliana through CRISPR/Cas9-mediated gene knockout and overexpression analysis. Gene editing of the homologous gene in Arabidopsis significantly reduced seed protein content in six lines, while two lines showed no obvious change. Overexpression of the AhSPC gene in Arabidopsis led to an increase in seed protein content in three transgenic lines, but some lines showed no significant change, or even a decrease. These inconsistent results might be attributed to functional redundancy, epigenetic modifications, resource competition, or feedback regulation mechanisms within the protein synthesis pathway. This study provides insights into the molecular mechanisms of peanut seed protein content regulation and offers a potential genetic target for molecular breeding.

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.