Supplemental Material

Figure S1. A BAC-based contig covering two copies of F3’H genes MdF3’HIIa and MdF3’HIIb in apple.

Figure S2. Developing a gene-tag for MdF3’HI gene using capillary electrophoresis method.

Figure S3. Amino acid sequence alignment of DFR fragments from different plants. The region marked in black indicates the substrate specificity determining region. The 134th asparagine and aspartic acid are marked by an arrowhead.

Figure S4. Expression profiles of MdFLS genes during different stages of fruit development in apple cvs. Red Delicious (red skin) and Gold Delicious (yellow skin) using real-time PCR. The cDNA templates are listed as follows: FTI: two weeks after pollination (WAP); FTII: 6 WAP; FTIII: 15 WAP; FTIV: 20 WAP; and FTV: 24 WAP. Transcriptional levels were normalized to expression of an apple actin gene. All data correspond to mean values of three biological replicates ± SD (bars).

Table S1: Primers for qPCR analysis of anthocyanin genes in apple and Arabidopsis

Species / Gene name / Gene identifier / Forward primer / Reverse primer
Apple / MdCHS / CN944824 / GGAGACAACTGGAGAAGGACTGGAA / CGACATTGATACTGGTGTCTTCA
MdCHI / CN946541 / GGGATAACCTCGCGGCCAAA / GCATCCATGCCGGAAGCTACAA
MdF3H / CN491664 / TGGAAGCTTGTGAGGACTGGGGT / CTCCTCCGATGGCAAATCAAAGA
MdDFR / AF117268 / GATAGGGTTTGAGTTCAAGTA / CGGCCGGAATCGGAATCAATC
MdLDOX / AF117269 / CCAAGTGAAGCGGGTTGTGCT / CAAAGCAGGCGGACAGGAGTAGC
MdUFGT / AF117267 / CCACCGCCCTTCCAAACACTCT / CACCCTTATGTTACGCGGCATGT
MdF3’HI / FJ919631 / ATGTCGGGAGGTGAGAAGCCCAATGTC / CGGCTCTTTGTAGGGTGAGCCCATAG
MdF3’HII / FJ919632 / CAGAAGGCCGACGAGTTCAAATCC / ATGTCCACGTGCTTCCCTTCG
MdFLS / AF119095 / AATGGGAGTGGAGTCTGTGG / AGTTGGAGCTGGCCTCAGTA
MdActin / CN938023 / TGACCGAATGAGCAAGGAAATTACT / TACTCAGCTTTGGCAATCCACATC
Arabidopsis / AtCHS / AT5G13930.1 / AACCCTGAGAACCATGTGCTTCAG / GATGCATGTGACGTTTCCGA
AtCHI / AT2G26310.1 / ACAGGAGCTTTGCTTTGTTGG / ATGGGAACACAGAGACGTTCC
AtF3H / AT3G51240.1 / AGGTTTTGTCTGAAGCTATGGGTC / TCAGTGTGACGCTTGAGTCCGA
AtF3’H / AT5G07990.1 / TCTAAATCCGTGGCCGAGCA / CATCGGTGTCCGTAAGGTGC
AtDFR / AT5G42800.1 / ACGTGGTTACTTTGTTCGTGCCA / CCGTTTATGGCATCATCGTAGC
AtLDOX / AT4G22880.2 / AAGAGCTCGAGAGCATCAACGA / GATGCCTTTTTCAGCTCCTCAA
AtUFGT / AT1G30530.1 / TGGCAGTTCTCGCGTTTTTCC / CGACGCGTTTGATCTTGCGGTG
AtActin / AT1G72150.1 / AGGAAGTTGCTGCTCCTGTC / ACCTTCTCAGCTACAACAACCGTC

Table S2. HPLC analysis of flavonoid contents in Arabidopsis seedlings grown on MS medium.

Seedlings / Flavonol (µg/g) / Anthocyanidin (µg/g) / Proanthocyanidin (µg/g)
Kaempferol / Quercetin / Pelargonidin / Cyanidin / Catechin / Epicatechin
Wild type / 17.8 / 6.6 / 17.6 / 0.6 / N/D / N/D
Mutant / 49.1 / N/D / N/D / N/D / N/D / N/D
MdF3’HI transgenic / 5.2 / 55.8 / 22.5 / 0.9 / N/D / N/D
MdF3’HII transgenic / 7.1 / 51.3 / 19.5 / 0.8 / N/D / N/D

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