Integrated metabolome and transcriptome analysis of anthocyanin accumulation during the color formation of bicolor flowers in Eustoma grandiflorum

•7 anthocyanins related to the bicolor flower formation of E. grandiflorum were identified.•The key structural genes and TFs related to flavonoid biosynthesis were screened.•EgCHS1 showed significantly higher expression in the upper tepals than in the tepal bases. The bicolor flowers of Eustoma grandiflorum have a special phenotype and high ornamental value. However, the coloration mechanism of these flowers has not been reported. We used metabolomics and transcriptomics to analyze the accumulation of secondary metabolites and the expression of differential genes in bicolor petals at different developmental stages, to elucidate the molecular mechanism underlying anthocyanin accumulation in these bicolor flowers of E. grandiflorum. The metabolomics analysis revealed that five kinds of anthocyanins in the upper pigmented petals at different developmental stages, and three kinds of anthocyanins were identified in the comparison between the pink upper tepals and the white bases at stage 3, with pelargonidin 3-glucoside 5-(6-coumaroyl) glucoside showing the most significant difference. The differentially expressed genes (DEGs) related to flavonoid biosynthesis in bicolor flowers were further analyzed by transcriptomics, and 25 structural genes and 5 MYB transcription factors (TFs) were screened, among which EgCHS1 showed significantly higher expression (approximately 21.5 times) in the upper tepals than in the tepal bases. Transcriptomic and metabolomic association analysis showed that the EgFLS2 and EgFLS3 genes were related to the largest quantity differentially accumulated metabolites (DAMs), and ferulate was associated with the largest quantity of DEGs. This study provides a basis for anthocyanin accumulation and coloration mechanism during the development of E. grandiflorum bicolor flowers. The results will be conducive for promoting artificial regulation of flower color and breeding new cultivars of E. grandiflorum.