Preliminary study on comparative non-targeted metabolomics analysis sheds light on the chemical diversity of citrus fruit pulps.

Citrus flavor and nutritional quality are closely tied to metabolite composition, yet comparative metabolic dissection of fruit pulp traits across citrus subspecies remains insufficient. Here, we applied non-targeted LC-MS/MS metabolomics to examine chemical diversity in the fruit pulp of three representative citrus varieties: Citrus reticulata 'Hongju 418', Citrus aurantium 'Changshan-huyou,' and Citrus junos 'Hunan Xiangcheng.' Through differential metabolite analysis, multivariate modeling, correlation network construction, and KEGG pathway enrichment, we characterized the extent and nature of metabolic divergence among these genotypes. PCA, PLS-DA, and OPLS-DA revealed distinct metabolic clusters, underscoring strong genotype-specific variation. More than 300 differentially expressed metabolites were identified, including flavonoid glycosides, organic acids, phenolic derivatives, and limonoids. Hongju 418 was enriched in flavonoid biosynthetic pathways, Xiangcheng accumulated higher concentrations of organic and amino acids, and Huyou displayed a unique hybrid profile marked by elevated fatty acid and purine metabolism. Correlation and KEGG analyses consolidated these observations, revealing coordinated pathway-level shifts that define subspecies-specific metabolic architectures. Collectively, this work deepens current understanding of citrus pulp chemotypes and provides a robust biochemical foundation for advances in citrus breeding, quality assessment, and functional product innovation.