Unveiling Genetic Determinants of Key Compounds in Citrus Leaves through Metabolite-Based Genome-Wide Association Study

A comprehensive metabolomics analysis was conducted on leaves from 239 genetically diverse citrus accessions selected from the Givaudan Citrus Variety Collection at the University of California, Riverside. Each accession was sampled with two biological replicates grafted onto distinct rootstocks, profiling 70 targeted and 713 untargeted metabolites. Principal Component Analysis (PCA) of these metabolites effectively separated major citrus subspecies, showing strong concordance with genomic classifications derived from 57,933 SNPs. A subset of 26 highly correlated metabolites (18 targeted, 8 untargeted) was identified, and pathway enrichment analysis using MetaboAnalyst revealed significant involvement in valine, leucine and isoleucine biosynthesis, among other pathways. Genome-wide association studies (GWAS) of these metabolites uncovered seven genomic loci. This led to the identification of candidate genes primarily enriched in pathways related to phytochelatin biosynthesis, metal ion response, and glutathione gamma-glutamylcysteinyltransferase activity. These findings reveal the genetic basis of variation in citrus leaf metabolites, offering new insights into their genetic architecture and laying the groundwork for metabolite-assisted breeding to enhance agronomic traits.