Chromosome-level genome assembly of Hamlin sweet orange and transcriptomic insights into MADS-box gene regulation of secondary fruit development

Abstract

Introduction The MADS-box gene family comprises key transcription factors that regulate plant growth and development, particularly flower and fruit morphogenesis. However, the evolutionary characteristics of this gene family in sweet orange (Citrus sinensis) and their specific regulatory roles in secondary fruit development remain unclear. This study aimed to systematically investigate these aspects by comparing cultivars with distinct fruit phenotypes. Methods Newhall navel orange (C. sinensis ‘Newhall’), which exhibits a typical secondary fruit phenotype, and Hamlin sweet orange (C. sinensis ‘Hamlin’), which lacks this trait, were used as experimental materials. A high-quality, chromosome-level de novo genome assembly was generated for Hamlin sweet orange. Comparative genomic analysis was performed to identify drivers of divergence between the two cultivars. Additionally, transcriptomic sequencing was conducted across bud, full bloom, and post-bloom stages, followed by weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with secondary fruit development. Results The Hamlin sweet orange genome assembly achieved an N50 of 32.89 Mb. Comparative genomics revealed that tandem duplication is a primary driver of divergence between the two cultivars. Gene family identification showed that Newhall possesses more MADS-box genes (139) than Hamlin (119). Transcriptomic and WGCNA analyses identified gene modules significantly correlated with secondary fruit development, pinpointing CSNSEP3 and CSNAG6 as key candidate regulatory genes. Discussion This study provides a high-quality reference genome for Hamlin sweet orange and offers valuable transcriptomic insights into the role of MADS-box genes in citrus differentiation and fruit morphogenesis. The identification of CSNSEP3 and CSNAG6 as critical regulators of secondary fruit development lays a theoretical foundation for molecular breeding programs targeting fruit morphology in citrus.