Functional analysis of a GWAS pleiotropic hotspot suggests an auxin biosynthesis gene ( AhPDS1 ), regulating pod development in peanut ( Arachis hypogaea L.)

SUMMARY Peanut productivity and quality improvement rely on understanding the genetic factors influencing pod and seed size. This study aims to identify genetic factors and regulatory mechanisms influencing pod and seed size in peanuts. Herein, a genome‐wide association study (GWAS) was conducted using 390 accessions from 15 peanut growing regions to analyze pod and seed traits across multiple planting seasons. A significant phenotypic variation was observed, with broad‐sense heritability ranging from 53.6 to 85.4%. Strong correlations between pod and seed traits further suggest potential for co‐selection in breeding efforts. A pleiotropic hotspot on chromosome B06 was strongly associated with six pod and seed traits. A peanut pod size regulator AhPDS1 ( PODSIZE‐1, Ahy_B06g085516 ) homolog of Arabidopsis thaliana YUCCA4 ( AtYUC4 , AT5G11320 ), involved in auxin biosynthesis, was selected as a candidate regulating pod and seed size. Quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) confirmed higher AhPDS1 expression in large pod as compared with the small pod genotypes. Subcellular localization showed AhPDS1 to be predominantly cytoplasmic, and GUS reporter assays indicated widespread expression in roots, stems, leaves, flowers, and pods, suggesting a broad functional role. Further overexpression of AhPDS1 in Arabidopsis and rice enhanced pod, seed, and grain sizes via the indole‐3‐pyruvic acid pathway in transgene lines. These findings highlight AhPDS1 as a potential target for peanut molecular breeding, offering opportunities to enhance pod size via auxin biosynthesis and support sustainable crop improvement.