Figure 2. Analysis of flavonoid accumulation and expression profiles of flavonoid biosynthesis genes during 5 leaf development stages in the Malus crabapple ever-red cultivar ‘Royalty’ and ever-green cultivar ‘Flame’.

(A) The five leaf developmental stages used for the analysis. (B) The content of the main flavonoids compounds in 5 leaf developmental stages of ‘Royalty’ and ‘Flame’. (C) Real-time PCR was used to analyze McMYB12a, McMYB12b, McCHS, McCHI, McF3H, McF3’H, McDFR, McANS, McUFGT, McFLS, McANR-1, McANR-2, McLAR-1, McLAR-2 expression patterns in the leaves of ‘Royalty’ and ‘Flame’. Malus 18 S (DQ341382) was used as the reference gene. S1 to S5 represents stage 1, 2, 3, 4 and 5 of leaf development. Error bars indicate the standard error of the mean ± SE of three replicate measurements. The expression levels and correlation of flavonoid regulatory and biosynthetic genes were calculated using CFX-Manager-3-1 following the manufacturer’s instructions (Bio-Rad). Scale bars = 1 cm. Different letters above the bars indicate significantly different values (P < 0.05) calculated using one-way analysis of variance (ANOVA) followed by a Tukey’s multiple range test.