Sucrose content dictates processing quality and consumer preference in edible peanuts, but its genetic control remains poorly characterized, limiting progress in breeding. In this study, using a breeding-informed F₂ population derived from a cross of high-sucrose germplasm JMT and elite cultivar Fushunsilihong (FSH) with normal-sucrose content, we integrated QTL mapping with functional genomics to decode the genetic architecture of sucrose accumulation. A major-effect locus (qSCB06) was precisely mapped to a 131-kb interval on chromosome B06 (148.03–148.16 Mb), accounting for 41.83% of the phenotypic variance (PVE). Comprehensive variant-to-function analysis identified five genes harboring exonic mutations, with Arahy.3URM83 validated as a key sucrose regulator through tissue-specific qRT-PCR profiling. To translate genomic discovery into breeding tools, we developed two cost-effective KASP markers (Arahy.06_115805462 and Arahy.06_148167024) that demonstrated 100% genotyping accuracy of F₂ individuals validated by SNP sequencing and 91.30% selection efficiency across 46 diverse cultivars. These markers enable rapid screening of sucrose-rich genotypes within 3 h, establishing a ready-to-implement molecular toolkit for expediting breeding of premium-quality peanuts. This work bridges gene discovery with field application, delivering both mechanistic insights and industrial solutions for flavor-driven peanut improvement.

Peanut (Arachis hypogaea L.) is an important oil and edible protein crop. Its fatty acid composition not only influences the quality of peanut oil but also impacts flavor, shelf life, and consumer health. Peanut oil is comprised of approximately 80% oleic acid (C18:1) and linoleic acid (C18:2), 10% palmitic acid (C16:0), and the remaining 10% includes stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0). To unravel the genetic foundation of fatty acid content and delve into QTL localization, high-density SNP microarrays were used to genotype the RIL population of ‘SunOleic 97R’ × ‘NC94022’. A genetic linkage map was constructed with 3,141 SNP markers, covering a total genetic distance of 3,051.81 cM. Sixty quantitative trait loci (QTLs) associated with fatty acids were distributed in 11 linkage groups, with phenotypic variance explained (PVE) ranging from 1.37 to 44.92%. Notably, the QTLs qFAT_A05.1 and qFAT_A08.1 are multiple-effect loci contributing to various fatty acid compositions. Moreover, 15 haplotypes for the QTLs qFAT_A05.1 and qFAT_A08.1 were identified through genotyping 178 peanut germplasms. Haplotype analysis in a natural population confirmed the close relationship of the QTLs with the contents of oil, oleic acid, lignoceric acid, palmitic acid and behenic acid. This study serves as a valuable reference for selecting improved peanut genotypes with superior oil quality and desirable fatty acid composition.