Validation of the up-regulated transcriptional pattern of the α-amylase gene from microarray data via qRT-PCR and enzyme activity analysis. (A) Up-regulated transcriptional pattern of Cit.17208.1.S1_at in microarray data, and this gene was annotated as α-amylase gene. (B) qRT-PCR analysis of α-amylase genes in M, RB, and SBT. Three α-amylase genes used for qRT-PCR analysis were from a previous report  and the NCBI. AMY, citrus sinensis alpha-amylase-like gene (accession number: XM_006473264); SD1, α-amylase gene (accession number: JN793456); SD2, α-amylase 3 gene (accession number: JN793457). Transcript levels are expressed relative to WT (wild type). (C) Enzyme activity analysis of α-amylase in wild-type M and M-33. α-amylase activity was expressed as mg maltose produced per gram tissue per minute. Columns and bars represent the means and ± SD, respectively (n = 3 replicate experiments). **Indicates that the values are significantly different compared with wild type at the significance level of P < 0.01. M, RB, and SBT represent Marsh grapefruit, Star Ruby grapefruit, and Sunburst mandarin, respectively. M-33 represents the ECM line of Marsh grapefruit.