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Bacterial wilt, caused by Ralstonia solanacearum , is a destructive disease with no effective chemical control, severely affecting global crop production. This study applied BSR-seq on 581 recombinant inbred lines (RILs), combined with linkage mapping, to identify resistance quantitative trait loci (QTL). Illumina sequencing yielded 189.6 Gb of data, identifying 70,035 high-quality SNPs from 55,840 genes. Two resistance loci were mapped on chromosome 12: a novel 1.11 Mb QTL and an adjacent 1.03 Mb region. Five CC-NBS-LRR-type resistance candidate genes were identified. The AhRRS6 alleles were cloned, and three allele-specific SNP markers were developed and validated across peanut breeding varieties. Additionally, 3,851 differentially expressed genes were detected, including key resistance-related genes. Transgenic AhRRS6y conferred strong resistance to R. solanacearum , while AhRRS6x caused susceptibility in both Nicotiana benthamiana and Arabidopsis thaliana . These alleles differentially regulated genes in HR, ETI, and PTI pathways, particularly affecting NbPDF1.2 and NbNDR1 . AhRRS6y expression reduced oxidative damage, indicated by lower malondialdehyde and higher ascorbate peroxidase activity. This work provides critical genetic resources for breeding bacterial wilt-resistant peanut varieties and enhances the mechanistic understanding of plant immune responses.
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