Identification of genes directly regulated by pleiotropic transcription factor duo1 (MYB125) in sex development in fig (Ficus carica L.)

Elucidating the molecular mechanisms underlying sex determination in plants is of strategic importance for both reproductive biology and agricultural productivity. Owing to its dioecious reproductive system, the fig (Ficus carica L.) constitutes an appropriate model species for investigating the molecular basis of sex development. In the present study, the transcription factor MYB domain protein 125 (DUO POLLEN1; DUO1), a central regulator of male gametophyte development, was characterized, and novel insights into the transcriptional regulation of sex determination were obtained by mapping DUO1 DNA-binding sites genome-wide. DUO1 binding regions were identified using antibody-free in vitro chromatin immunoprecipitation sequencing (in vitro ChIP-seq), and high-depth next-generation sequencing data were subjected to comprehensive bioinformatic analyses. The DUO1 ortholog was identified through BLAST searches, R2R3-MYB domain architecture analysis, and phylogenetic inference. İn vitro ChIP-seq analyses conducted in male and female fig cultivars revealed numerous DUO1 binding sites across the genome. Functional annotation based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated significant enrichment of biological processes associated particularly with anther and pollen development. In accordance with this functional profile, ten candidate male marker genes were selected, and DUO1–DNA interactions within the promoters of these genes were validated by electrophoretic mobility shift assay (EMSA). Collectively, this study provides strong genomic and regulomic evidence that DUO1 establishes a distinctive transcriptional network comprising target genes involved in male gametophyte development in fig. These findings offer a methodological framework that can be directly integrated into modern breeding programs by enabling the development of molecular markers for early sex identification and for the assessment of male reproductive quality.