Abstract:Anthocyanins are plant secondary metabolites with important physiological activities, and deciphering the mechanism of pigment formation has become one of the most important research hotspots. In this study, the peanut varieties including Zizhenzhu (purple testa), Hongzhenzhu (red testa), G110 (pink testa) and Baizhenzhu (white testa) were analyzed using RNA-Seq and liquid chromatography tandem mass spectrometry (LC-MS/MS), by using testa samples harvested at 30 and 45 days after flowering (30 DAF and 45 DAF), respectively. RNA-Seq revealed 32805 differentially expressed genes (DEGs), which were enriched in different number of pathways using KEGG analysis. GO analysis revealed 34, 21 and 19 DEGs which were enriched to the pathways of oxidation-reduction process, anthocyanin-containing compound biosynthetic process and Flavonoid biosynthetic process, respectively. LC-MS/MS analysis showed types of metabolites including proanthocyanidins, petunidin, paeoniflorin, malvadin, delphinium, cyanidin and their derivatives. Procyanidins A1, A2, B2, B3, delphinium and cyanidin were significantly up-regulated in each comparison group, with a variation of 5.82-19.52 on fold. Two pathways consisting of anthocyanin biosynthesis and flavonoid biosynthesis were enriched. The transcriptomic-metabolomics combined analysis showed that flavonoid biosynthesis is the key synthesis pathway for testa color formation, and delphinidin and cyanidin are the main differential metabolites. Out of 20 important genes, each), qRT-PCR analysis showed that PAL, 4CL, IF7MAT, CHI, F3H, DFR, LAR and LDOX were significantly up-regulated; C-CoA and FLS were observably down-regulated at 30 DAF; PAL, HCT-1 and DFR were significantly up-regulated; CHS, C-CoA and FLS were observably down-regulated at 45 DAF. Collectively, these results laid a theoretical foundation for future deciphering the molecular mechanism of peanut testa anthocyanin synthesis, valuable in breeding for peanut varieties with enriched anthocyanin.