Supplementary Table 1 Sequences of primers used in 3’RACE and TAIL-PCR

Gene / Sequence(5’-3’) / Annealing temperature / Function
PAL / CAGAACGGTGCTGTGGAGTC
TCGCAGAATCTTGAGGGGTG / 54 / 3’RACE
TCGCAGAATCTTGAGGGGTG
CCTGCTCATGCAACTTCTTA
GACACGGTGAGCCAAGTCG / 55 / TAIL-PCR
CHS / AGGGCTTACATTTCACCTTCT
CTACTTTCGTATCACCAACAG / 56 / 3’RACE
CAGAAGAGCGAGTTCCAGTC
TGTGCTCGCTGTTGGTGATACG
GCAGCACAAACCATTCTTCC / 55 / TAIL-PCR
CHI / GGTAAGACCGCCGAGGAGTTGT
TTACAGGTCCGTTTGAGAAGTT / 54 / 3’RACE
CTCAAACGGACCTGTAACGATGT
CCGTCAGTGGCAGTATCGTTGT
AGTTTGTTTTCTATCACCGCATTTG / 58 / TAIL-PCR
F3H / CAGTGGTGAACTCAAACAGTAGCA
CTACTCGGGATGATGGGAAA / 56 / 3’RACE
CTTCTCTCCCTCCCTCACACA
GGCTATGGACAGCCTGCTAC
GGCATGTGTGGACATGGACCC / 52 / TAIL-PCR
DFR / CCCCGAGTACAATATACCCACAA
ATGTTGAATTTTGCCGCTTC / 55 / 3’RACE
GAACTCAAACCCTATCTCCCTCAA
GAATGGATGGCGTGAGAAAT
GACCCCACGAATCAGAAGAAGG / 56 / TAIL-PCR
ANS / GCACTCACCTTCATCCTCCAC
GACCTTCCCATTGAGCAGAA / 54 / 3’RACE
TGCTCAAAATCTCAAGTGTGTCC
CAAGTCACGCTTGTCCTCTG
ATTCGAGCTCGGTACCCGGGG / 55 / TAIL-PCR
UFGT / CCCCGTAACAAACGACCTAA
AGAACCTTGATCTGATCCAAG / 55 / 3’RACE
TTGCTAGCAGCTCCTTCTCC
TGGGTTATTAGAGATAAGGTCG
CTCCACATTTACCCCTATCTCC / 60 / TAIL-PCR
3’-RACE / TCCACGATTGATGTCACGATAGG
TGTGATGCTCCACGATTGATGTC / 50~60 / 3’RACE
TAIL-
PCR / NGTCGASWGANAWGAA
WGTGNAGWANCANAGA
AGWGNAGWANCAWAGG
GTNCGASWCANAWGTT / 48~55 / TAIL-PCR

Supplementary Table 2 The full-length cDNA and deduced protein characteristics

for anthocyanidin biosynthesis genes

Gene / Full-length cDNA
(bp) / Polypeptide / Calculated molecular mass(kDa) / Predicted isoelectric point
PAL / 2402bp / 720 / 78.12kDa / 6.32
CHS / 1173bp / 389 / 42.26kDa / 5.77
CHI / 748bp / 219 / 23.41kDa / 5.60
DFR / 1044bp / 347 / 38.88kDa / 5.87
F3H / 1095bp / 364 / 40.86kDa / 5.29
ANS / 1074bp / 357 / 40.33kDa / 5.62
UFGT / 1404bp / 363 / 39.56kDa / 6.79

Supplementary Table 3 Homology search result in GenBank (NCBI) between the amino acid sequences cloned in this study and other plant species of cDNA sequences

Deduced protein / Species / Top Blast Match / Homology（%）
PAL / Pyrus communis / ABB70117.2 / 97%
Vitis vinifera / ABM67591.1 / 95%
CHS / Malus x domestica / AAY45748.1 / 100%
Fragaria x ananassa / BAE17124.1 / 94%
CHI / Malus x domestica / AAM12891.1 / 91%
Prunus avium / ADZ54781.1 / 83%
F3H / Malus x domestica / BAB92997.1 / 97%
Prunus avium / ADZ54782.1 / 93%
DFR / Malus x domestica / AAO39816.1 / 99%
Vitis amurensis / ACN82380.1 / 82%
ANS / Malus x domestica / BAB92998.1 / 100%
Fragaria x ananassa / AAU12368.1 / 99%
UFGT / Malus x domestica / BAK19404.1 / 90%
Prunus avium / ADZ54786.1 / 97%
MYB10 / Pyrus communis / EU153575.1 / 99%
Pyrus pyrifolia / HM585181.1 / 98%
bHLH / Pyrus pyrifolia / DQ266451.1 / 98%
Malus x domestica / DQ266451.1 / 97%
WD40 / Pyrus pyrifolia / HQ641374.1 / 99%
Malus x domestica / GU173814.1 / 99%

Supplementary Table 4 Correlations between anthocyanin content and the anthocyanin biosynthesis genes

RED / anthocyanin / MYB10 / BHLH / WD40
anthocyanin / Pearson Correlation / 1 / 0.859** / 0.917** / 0-.951**
Sig. (2-tailed) / 0 / 0 / 0
N / 12 / 12 / 12 / 12
MYB10 / Pearson Correlation / 0.859** / 1 / 0.703* / -0.801**
Sig. (2-tailed) / 0 / 0.011 / 0.002
N / 12 / 12 / 12 / 12
BHLH / Pearson Correlation / 0.917** / 0.703* / 1 / -0.847**
Sig. (2-tailed) / 0 / 0.011 / 0.001
N / 12 / 12 / 12 / 12
WD40 / Pearson Correlation / -.951** / -0.801** / -0.847** / 1
Sig. (2-tailed) / 0 / 0.002 / 0.001
N / 12 / 12 / 12 / 12

**. Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

GREEN / anthocyanin / MYB10 / BHLH / WD40
anthocyanin / Pearson Correlation / 1 / 0.221 / 0.057 / -0.456
Sig. (2-tailed) / 0.489 / 0.86 / 0.136
N / 12 / 12 / 12 / 12
MYB10 / Pearson Correlation / 0.221 / 1 / 0.755** / 0.676*
Sig. (2-tailed) / 0.489 / 0.005 / 0.016
N / 12 / 12 / 12 / 12
BHLH / Pearson Correlation / 0.057 / 0.755** / 1 / 0.48
Sig. (2-tailed) / 0.86 / 0.005 / 0.114
N / 12 / 12 / 12 / 12
WD40 / Pearson Correlation / -0.456 / 0.676* / 0.48 / 1
Sig. (2-tailed) / 0.136 / 0.016 / 0.114
N / 12 / 12 / 12 / 12

**. Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

Supplementary figure 1 cDNA cloning and agarose gel electrophoresis of anthocyanin biosynthesis structural genes in the ‘Early red Doyenne du Comice’ strain (Left) and the ‘Green Doyenne du Comice’ strain (Right).

Supplementary figure 2 cDNA cloning and agarose gel electrophoresis of anthocyanin biosynthesis transcription factors in the ‘Early red Doyenne du Comice’ strain (Left) and the ‘Green Doyenne du Comice’ strain (Right).