Identification of Key Pathways and Candidate Genes Controlling Organ Size Through Transcriptome and Weighted Gene Co-Expression Network Analyses in Navel Orange Plants (Citrus sinensis)

Background/Objectives: Organ size is a critical target trait in fruit-tree breeding programs, as it significantly impacts the economic value of plants by influencing their biomass, yield, and quality. Understanding the molecular mechanisms underlying organ size in citrus is essential for breeding new cultivars with superior fruit quality. Methods: In this study, we investigated the regulatory network involved in organ size using the Citrus sinensis ‘Newhall’ navel orange variety and its large-organ mutant, ‘M25’. Results: Ploidy analysis indicated that the organ enlargement observed in ‘M25’ was not attributable to changes in chromosome ploidy. Furthermore, RNA sequencing of tender leaves and young fruits from both ‘M25’ and ‘Newhall’ oranges identified 1817 and 1605 differentially expressed genes (DEGs), respectively. Functional enrichment analysis revealed that these DEGs were enriched in pathways associated with organ size regulation, including those related to cell division, DNA replication, protein biosynthesis, plant hormone signal transduction, and cell wall metabolism. Weighted gene co-expression network analysis identified the grey 60 and orange modules as the key modules influencing organ enlargement; from these modules, we identified 51 and 35 hub genes, respectively. Combined homologous function annotation and expression analysis identified four transcription-factor-encoding hub genes (Cs_ont_6g005380, Cs_ont_8g025330, Cs_ont_9g019400, and Cs_ont_9g008010) as candidate genes potentially related to organ size. Conclusions: Among these, Cs_ont_8g025330 (CsMYB73) was inferred to be the key gene influencing organ size through auxin and cytokinin regulation. These findings lay the foundation for further investigations of the regulatory mechanism of organ size in navel orange varieties.