Fig. 3
GWAS identification of candidate genes associate with CNglcs content. a Manhattan plots for total CNglcs content in 241 accessions using MLMM. The black dashed lines indicate the significance threshold (p value = 2.0 × 10.−5) and black arrow indicates the significant GWAS peak. b Gene model and SNPs of LjMTR. Exons, introns and promote are represented by blue boxes, black lines and red line, respectively. The non-synonymous SNP is marked by a black arrow. Hap.G, haplotype G (GG); Hap.S, haplotype S (GC). c Box plot of total CNglcs content, the white box and gray box represent Hap.G (GG) and Hap.S (GC), respectively. The significance of difference was derived with two-tailed t-test (*P < 0.05). d–e Relative expression of LjMTR and LjZCD in different accessions carrying Hap.G and Hap.S, respectively. The white bar and gray bar represent Hap.G and Hap.S, respectively. f Schematic diagram of reporter and effector in Arabidopsis protoplast transactivation assays. Reporter is the fusion of the CYP79D3, CYP736A2, and UGT85K3 promoter with firefly luciferase gene (LUC). The LjMTR, site mutated LjMTR-D230 and LjZCD fused with the CaMV 35S promoter respectively to from effectors. NOS, the transcriptional terminator of the nopaline synthase gene from Agrobacterium tumefaciens. g Luciferase assays of LjMTR, and site mutated LjMTR-D230 regulate the expression of CYP79D3, CYP736A2, and UGT85K3. The empty vector was used for control. Values represent the mean ± SE of triplicate experiments. Significant differences between values are indicated with different letters (*, P < 0.05; **, P < 0.005; ***, P < 0.0001 one-way ANOVA)