Nut kernel color is a crucial quality indicator affecting consumers' first impression of the product. In the postharvest stage, nut kernels (e.g., pecans) gradually darken with increasing blue/red colors, which have a negative impact on consumers' preference. While growing evidence suggests that plant phenolics and their derivatives are linked to pecan kernel color, the compounds (biomarkers) responsible for kernel color stability during storage remain elusive. We hypothesized that: (1) the phenotype of pecan color exhibits notable variation among different cultivars during storage; and (2) this variation is attributed to changes in metabolites and their combination effects in the stage of phenolic biosynthetic pathways related to color formation. Here, pathway-based metabolomics with machine learning (ML) algorithms were employed to identify key metabolites of postharvest pecan color stability. Nine pecan cultivars with different color stabilities were selected, and the color of each cultivar was measured over a 6-month storage period (0, 1, 2, 3, and 6 months). Metabolites in phenylpropanoid, flavonoid, and anthocyanin biosynthetic pathways were analyzed in the outer layer of the kernel (testa) using liquid chromatography–mass spectrometry. Different ML models were compared to find relevant biomarkers of pecan color phenotypes. Lasso regression and support vector machine (linear kernel) in conjunction with recursive feature elimination were chosen as the most effective models to select biomarkers that predict the kernel color of pecans. Results revealed twenty marker compounds (flavonoid precursors, flavonoids, anthocyanidins, and anthocyanins) within phenylpropanoid, flavonoid, and anthocyanin biosynthetic pathways, and their roles in pecan color phenotypes during storage. Our findings provide a foundation for future research in the area and will help select genes/proteins for the breeding of pecans with stable and desirable kernel color. The collected information on biomarkers and their effects will also help set up strategies for the quality control of pecans after harvest.
