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Sweet sorghum (Sorghum bicolor) is one of the most important cereal crops in the world with colorful seeds. To study the diversity and cultivar-specificity of phytochemicals in sweet sorghum seeds, widely targeted metabolomics was used to analyze the metabolic profiles of the white, red, and purple seeds from three sweet sorghum cultivars Z6, Z27, and HC4. We identified 651 metabolites that were divided into 24 categories, including fatty acids, glycerolipids, flavonoids, benzoic acid derivatives, anthocyanins, and nucleotides and its derivatives. Among them, 217 metabolites were selected as significantly differential metabolites which could be related to the seed color by clustering analysis, principal component analysis (PCA), and orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA). A significant difference was shown between the red seed and purple seed samples, Z27 and HC4, in which 106 were downregulated and 111 were upregulated. The result indicated that 240 metabolites were significantly different, which could be related to the purple color with 58 metabolites downregulated and 182 metabolites upregulated. And 199 metabolites might be involved in the red phenotype with 54 downregulated and 135 upregulated. There were 45 metabolites that were common to all three cultivars, while cyanidin O-malonyl-malonyl hexoside, cyanidin O-acetylhexoside, and cyanidin 3-O-glucosyl-malonylglucoside were significantly upregulated red seeds, which could be the basis for the variety of seed colors. Generally, our findings provide a comprehensive comparison of the metabolites between the three phenotypes of S. bicolor and an interpretation of phenotypic differences from the point of metabolomics.

Metabolite Profiling of Sorghum Seeds of Different Colors from Different Sweet Sorghum Cultivars Using a Widely Targeted Metabolomics Approach