Abstract:Peanuts are an important product as part of the world's cereals, oils and foods. The testa colors of peanuts are white, red, purple, pink, and variegated. Variegated peanut testa are unique and can be distinguished from other types. In this study, the variegated testa peanut VG-02 was adopted as the research material. The study showed that the differentially expressed miRNA-enriched metabolic pathways related to the color synthesis included phenylpropane biosynthesis, flavonoid biosynthesis, isoflavone biosynthesis and rhythm-plant. MiRNA sequencing results showed that 86 miRNAs were differentially expressed of which 20 miRNAs were related to the color synthesis in variegated testa peanut. Twenty differentially expressed miRNAs related to anthocyanin synthesis in variegated testa peanut including miR_8, miR_50, miR_51 and miR_239-x, that jointly targeted anthocyanins, anthocyanidins and IFS target gene. Five miRNAs were found to targete structural genes in the anthocyanin biosynthesis: miR_398-x which regulates CHS target genes, miR_482 which regulates 4CL target genes, miR_266 and miR_182 which regulate F3'H target genes and miR_5 which regulates the anthocyanin 3-O-glucoside target gene. MiR858-y is a miRNA targeting anthocyanin biosynthesis regulation gene, which targets and regulats MYB2 and MYB3. MiR_10, miR_15, miR_61, miR_72, miR_102, miR_116, miR_123, miR_193, miR_256 and miR_862-z target CYP450 target genes.The joined analysis of KEGG metabolic pathways by miRNA sequencing and transcriptome analysis indicated that flavonoid biosynthesis is the most direct metabolic pathway for the synthesis of testa variegation. This study further our understanding of the molecular mechanisms of anthocyanin synthesis in variegated testa peanut.