Structural variations, gene polymorphism and expression reveal major candidate genes associated with pod and seed size variation during peanut (Arachis hypogaea L.) domestication

Pod size is a key agronomic trait in peanut (Arachis hypogaea) that underwent strong selection during domestication. In a previous study, we fine-mapped a quantitative trait locus (QTL) for pod and seed size to a 168.37 kb region on chromosome A07. Here, we integrated structural variation analysis, gene sequence comparison, and transcriptomic profiling to refine candidate genes and uncover molecular mechanisms underlying domestication-related differences in pod size. Comparative genomic analyses among Arachis duranensis, A. monticola, and four cultivated varieties (Fleur11, Tifrunner, Shitouqi, Fuhuasheng) revealed multiple structural variations in the QTL interval, including a ~ 25 kb inversion between the wild species (A. duranensis and A. monticola) and all cultivated genotypes. This inversion altered the orientation of a candidate gene, Aradu.DN3DB/Arahy.5EZV1I (SAP), which also carried a non-synonymous SNP distinguishing wild and cultivated lines. RNA-Seq profiling of two parental lines (Fleur11 and 12CS_091) and two near-isogenic lines (NILs) at 20 and 40 days after flowering (DAF20, DAF40) identified two differentially expressed genes in the QTL region: Aradu.SFU0J/Arahy.VEUG4Z (phytochromobilin synthase) and gene.15763 (mitochondrial ribosomal protein), both showing higher expression in large-pod genotypes. GO enrichment analysis revealed contrasting strategies between pod size groups: small-pod genotypes emphasized early maturation and storage metabolism, while large-pod genotypes maintained prolonged growth through hormonal signaling and cell division, supporting greater pod expansion. This study identifies three promising candidate genes for seed and pod size variation during peanut domestication: Aradu.DN3DB/Arahy.5EZV1I, gene.15763, and Aradu.SFU0J/Arahy.VEUG4Z. The ~25 kb inversion affecting Aradu.DN3DB/Arahy.5EZV1I and the expression differences in Aradu.SFU0J/Arahy.VEUG4Z between large- and small-pod genotypes suggest structural and transcriptional mechanisms contribute to phenotypic variation. These findings advance understanding of domestication-related traits in peanut and provide a foundation for functional validation, which could guide breeding strategies to optimize pod and seed size.