ABSTRACT: Background The first branch angle (FBA) is a critical trait influencing plant architecture, yield, and mechanized harvesting efficiency in pepper (Capsicum annuum L.). However, the genes and regulatory processes involved remain largely unclear. Results The phenotypic evaluation of 220 pepper accessions revealed significant genetic variation in FBA, with a broad-sense heritability of 93.26%, indicating a strong genetic foundation and making it a stable trait for selection. A genome-wide association study (GWAS) with mixed linear model and FarmCPU detected 25 significant SNPs related to FBA. Several candidate genes were identified, encompassing components of brassinosteroid signaling, gibberellin signaling, serine/threonine-protein kinase signaling, and cell wall modification. To functionally contextualize these genetic findings, two accessions with divergent FBAs (compact B010 and loose B003) were selected for cytological and hormonal analyses. The paraffin sections stained with Periodic Acid-Schiff showed that the compact B010 exhibits more starch granules in endodermal amyloplasts, while the loose B003 has larger cell size on the adaxial side. Hormonal analysis based on HPLC-MS/MS revealed higher auxin levels in the compact B010 and higher gibberellin, cytokinin, brassinosteroid, and strigolactone levels in the loose B003. These results suggest a mechanism whereby brassinosteroid/gibberellin-mediated cell expansion, potentially through the action of cell wall modification genes, drives the loose architecture of B003. Furthermore, RNA-Seq and qRT-PCR confirmed the coordinated regulation of the key phytohormone pathways (auxin signaling; cytokinin, gibberellin, and brassinosteroid biosynthesis) and serine/threonine-protein kinase signaling. Therefore, our work integrates multi-layered analyses to propose a regulatory framework for FBA in pepper: genetic loci associate with alterations in phytohormone regulation, kinase activity, and cellular traits, which orchestrate the transcriptomic, hormonal, and cytological changes that collectively determine branch architecture.
