Betalains are tyrosine-derived pigments that occur solely in one plant order,the Caryophyllales,where they largely replace the anthocyanins in a mutually exclusive manner.In this study,we conducted multi-species transcriptome and metabolic profiling in Mirabilisjalapa and additional betalain-producing species to identify candidate genes possibly involved in betalain biosynthesis.Among the candidates identified,betalainrelated cytochrome P450 and glucosyltransferase-type genes,which catalyze tyrosine hydroxylation or (hydroxy)cinnamoyl-glucose formation,respectively,were further functionally characterized.We detected the expression of genes in the flavonoid/anthocyanin biosynthetic pathways as well as their metabolite intermediates in betalain-accumulating M.jalapa flowers,and found that the anthocyanin-related gene ANTHOCYANIDIN SYNTHASE (MjANS) is highly expressed in the betalain-accumulating petals.However,it appears that MjANS contains a significant deletion in a region spanning the corresponding enzyme active site.These findings provide novel insights into betalain biosynthesis and a possible explanation for how anthocyanins have been lost in this plant species.Our study also implies a complex,non-uniform history for the loss of anthocyanin production across betalain producers,previously assumed to be strictly due to diminished expression of anthocyanin-related genes.