Multi-omics analysis of genetic differences between Guangchenpi and Huajuhong

• Differences in evolutionary and differentiation relationships between citrus reticulata ‘ Chachiensis ’ and citrus grandis tomentosa . • Differences in metabolic pathways and relative expression levels of differentially expressed genes between citrus reticulata ‘ Chachiensis ’ and citrus grandis tomentosa . • Differences of five major metabolites in the peels of citrus reticulata ‘ Chachiensis ’ and citrus grandis tomentosa . Guangchenpi and Huajuhong are valuable Chinese medicinal herbs mainly derived from Citrus reticulata ‘Chachiensis’ (CZG) and Citrus grandis Tomentosa (HZY). However, genetic differences between Guangchenpi and Huajuhong have not been analysed, which hinders a thorough understanding and optimisation of their efficacy and quality control. Herein, genome, transcriptome and metabolome multi-omics analysis was employed to compare genetic differences between CZG and HZY. CZG contained more genes, especially those related to metabolic activity. The differentiation time of pomelo-associated genera with closer evolutionary relatedness to HZY was earlier than that of citrus-associated genera with closer evolutionary relatedness to CZG. CZG and HZY had more genes associated with flavonoids and terpenoids upregulated in peel than other tissues. Widely-targeted and volatile metabolome analysis revealed that changes in flavonoids and terpenoids were consistent with the expression patterns of related pathway genes. The five major categories of metabolites in the peel of CZG and HZY, namely flavonoids, lignins, coumarins, terpenoids and alkaloids, were compared. Differences in these metabolites largely explained the differences in medicinal properties between CZG and HZY. This study provides insights for the cultivation and development of new traditional Chinese medicine products in the future.