A large sensory and multi-omics evaluation unraveled chemical and genetic basis of orange flavor

Sweet orange (Citrus sinensis) exhibits limited genetic diversity and high susceptibility to Huanglongbing (HLB). New HLB-tolerant orange-like hybrids are promising alternatives. However, the genetic control of key flavor compounds in oranges remains unknown. Evaluating 179 juice samples, including oranges, mandarins, Poncirus trifoliata and hybrids, distinct volatile compositions were found. A random forest model predicted untrained samples with 78% accuracy and identified 26 compounds crucial for orange flavor. Notably, seven esters--methyl hexanoate, ethyl hexanoate, ethyl 3-hydroxyhexanoate, ethyl octanoate, methyl butanoate, ethyl butanoate, and ethyl 2-methylbutanoate--differentiated orange from mandarin. Cluster analysis showed six esters with shared genetic control. Differential gene expression analysis identified CsAAT1, an alcohol acyltransferase responsible for ester production in orange. Its activity was validated through overexpression assays. A SNP-based DNA marker in the CDS region accurately predicted phenotypes. This study enhances our understanding of orange flavor compounds, their biosynthetic pathways, and expands breeding options for orange-like cultivars.