Fig. 4

Functional annotation of genes responsible for variation in anthocyanin contents and SR endothelium color. A-B Manhattan plot displaying the GWAS results of cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside. C Manhattan plot displaying the GWAS results for SR endothelium color. D Phenotypes of cassava SR with white or red endothelium. E Gene model of Sc02g007130 (MeMYB4). The lead SNP 2:6017365 is indicated by a vertical arrow. The causative SNP 2:6158256, which located in the promoter of MeMYB4, is indicated by a red star. F Expression level of MeMYB4 in leaf, root, stem, and among seven developmental stages (S1–S7) of storage root. G Phylogenetic tree of MeMYB4 and MYB genes from other species. Bootstrap values are indicated at each node. H Comparison of activities between C-containing and T-containing promoter regions (1000 bp upstream of ATG) in dual luciferase assay. Each sample contains seven biological replicates. I-J Boxplot displaying the relative contents of cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside based on the single likely causative SNP 2:6158256. K The percentage of SR endothelium color attributable to the causative SNP 2:6158256. L-M Pearson correlations between the expression of MeMYB4 and the content of cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside in SR of 22 cassava cultivars, respectively. N Reduced transcript expression of MeMYB4 in MeMYB4-silenced cassava plants (pTRV-MeMYB4-1 and pTRV-MeMYB4-2) compared with the control (pTRV) in leaf. Bars indicate mean ± SD, n = 3. O-P Reduced levels of cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside in MeMYB4-silenced cassava plants (pTRV-MeMYB4-1 and pTRV-MeMYB4-2) compared with the control (pTRV) in leaf. Bars indicate mean ± SD, n = 3. Q Growth of yeast cells co-transformed with pHis2.1-ProMeCHI/pGADT7-MeMYB4, pHis2.1-ProMeF3H/pGADT7-MeMYB4, or the positive control (pHis2.1-P53/pGADT7-Rec2-53). R Reduced transcripts of MeCHI and MeF3H in MeMYB4-silenced cassava plants (pTRV-MeMYB4-1 and pTRV-MeMYB4-2) compared with the control (pTRV) in leaf. Bars indicate mean ± SD, n = 3. Asterisk symbols (**) in this figure indicate a significant difference at P < 0.01 by the Student’s t-test