Transcriptomic and co-expression network analysis reveals coordinated multi-pathway regulation of blueberry fruit ripening

Abstract Blueberries are valued for their colour, flavour, and high polyphenol content, particularly anthocyanins. Understanding the transcriptional regulation underlying fruit development and ripening is essential for improving fruit quality and nutritional value. This study aimed to characterise gene expression changes across key developmental stages in highbush blueberry and identify the molecular pathways associated with ripening. A two-year transcriptomic analysis was conducted on ‘Chandler’ highbush blueberry ( Vaccinium corymbosum ) at three developmental stages: green (S1), expansion (S2), and fully ripe (S3). Messenger RNA libraries were sequenced, generating approximately 25 million paired-end reads per sample. Differential expression analysis identified 11,470 genes that showed significant differential expression between the green (S1) and fully ripe (S3) stages in both years, indicating extensive transcriptional reprogramming during ripening, whereas earlier transitions involved fewer changes. Functional enrichment revealed that early development was associated with cell wall modification processes, including pectate lyase and cellulase activity, while ripening stages were enriched for phosphatase and hydrolase activities. Weighted co-expression network analysis identified modules related to flavonoid biosynthesis, cell wall remodelling, antioxidant processes, signalling, and sugar transport. Key genes, including CYP75A1 and UGT90A1 (flavonoid biosynthesis), NCED1 (abscisic acid biosynthesis), XTH28 and BGAL8 (cell wall enzymes), PER42 and CuAOγ1 (antioxidant enzymes), and TORL3 (microtubule-associated protein), presented strong stage-specific expression patterns and high network connectivity. These patterns were consistent across both growing seasons. This study provides a systems-level view of blueberry fruit development, highlighting coordinated transcriptional changes across multiple biological pathways during ripening. The identification of conserved gene expression patterns and key regulatory candidates offers valuable targets for improving fruit quality and health-promoting traits in blueberry.