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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.