Supplementary Table 1 Primers used for quantitative PCR
Gene
Forward primer
Reverse primer
OsFER
AGGGATTCGCCAAATTCTTC
CCTTTCTCAGGATGGTCGAA
NAS1
AGCGCGGTTGAGAAGGCAGAA
GGCATGTTCCTCGTTTACACTGGC
NAS2
CGCCAAGAGGCTATCCGTCTGA
GATCAATGCCCAGGCGCCTA
NAS3
CGAGTGTTCGACTGATCACC
CCGGTGATCTTCTCAACCAA
NAAT1
TGCTGTGGCAGATCATTTGT
TTGGGTAGCCTGGTCTAGGA
SoyFERH1
GGGATTTGCCAAGTTCTTCA
TACAATGCATCCCCCTTTTC
YSL15
TGATCATAGCCTACGCGCTCGG
GCGATGAAGCAGCCAATGCC
YSL18
GACAAGCGAAGAGGCAAGTC
CGATTCCCACATAAGTTCCG
FRO1
GGCCACCAAGCAGTCCCTCTTT
CGAAAGGCCCGTCCACAAGA
FRO2
TCCCTGTTGCCGCTCATTGG
CCCGGGGACGTTTGCAACTT
Supplementary Table 2 Grain quality assessment of transgenic IR64 lines
Sample ID
DESIGNATION
IR64 control
Cooking traits
AC
GT
GC
Low
HI/I
Soft
PC
(%)
12.2
Low
I
Soft
Intermediate
I
Intermediate
HI/I
Milling potential
score
DB
C
1
1
11.6
1
1
Soft
12.5
1
2
Soft
12.6
1
2
Single transformation
IR64-GLUB4:: SoyFERH1018B-003
IR64- GLUB4::OsFER1C-088A006
IR64-GLUB1:: SoyFERH2176A-002
Co-transformation
IR64- GLUB1:: SoyFERH1Low
HI/I
Soft
12.5
1
2
028A-109
IR64- GLUB1:: SoyFERH1Intermediate
HI/I
Soft
10.1
1
2
072A-094
IR64-GLUB1:: SoyFERH1-37A- Intermediate
I
Soft
11.7
1
2
129
AC = amylose content: waxy (0-2%), very low (3-9%), low (10-19%), intermediate (20-25%), high
(>25%); GT = gelatinization temperature: I = intermediate (70-72 C), HI = high intermediate (73-74 C),
results are from six replicates; GC = gel consistency scored as 1) very flaky rice with hard gel consistency
(length of gel, 40 mm or less); 2) flaky rice with medium gel consistency (length of gel, 41 to 60 mm); and
3) soft rice with soft gel consistency (length of gel, more than 61 mm), results are from two replicates; PC
= protein content, with range of 8 to 14 considered as normal or marketable; DB = degree of breakage and
C = chalkiness score, both classified using the following scale: 1 = mostly whole translucent grains; 2 =
few broken/chalky grains, and 3 = >70% of grains are broken; >50% chalky grains
Supplementary Table 3 Comparison of average iron concentration of IR64 transgenic plants (single
transformants) with ferritin genes driven by GLUB1 versus GLUB4 promoter. Average iron concentration
for each promoter were obtained from 8 transgenic plants
Promoter
T2 ICP (mg kg-1)
T3 ICP (mg kg-1)
GLUB1
5.63 ± 0.67
4.60 ± 1.20
GLUB4
6.21 ± 0.74
5.03 ± 1.80
IR64:GLUB1:GUS
IR64:GLUB4:GUS
IR64:GLB:GUS
IR64:35S:GUS
IR64
Dough (15 DAF)
Mature (30 DAF)
Supplementary Fig 1 Histochemical GUS staining of dough (left) and mature seeds (right) of transgenic
IR64 plants with different promoter:GUS constructs
M
1 2
3
4
5
6 7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 WT
kb
8.5
7.4
6.1
4.8
3.6
2.7
Supplementary Fig. 2 Southern blot analysis of transgenic IR64 with SoyFERH1 driven by GLUB1
promoter. Genomic DNA from each sample digested with EcoRI, hybridized with a PCR-labeled probe
containing 928-bp fragment of SoyFERH1 gene. M = Dig-labeled marker VII, WT = wild-type IR64, 1-28
= T0 events with SoyFERH1gene
Supplementary Fig. 3 Screenhouse evaluation of phenotype of different T1 transgenic lines
Fe mg kg -1
9
8
7
6
5
4
3
2
1
0
T2 ICP
T3 ICP
R
FE
s
O
1C
R
FE
y
So
H1
R
FE
y
So
H2
Ferritin Gene
Supplementary Fig. 4 Comparison of iron concentration of IR64 transgenic plants with different ferritin
genes
[GENETYX-MAC: Multiple Alignment]
Date
: 2013.03.08
Transit peptide
OsFER1
1 -----MLPPRVAPAAAAAAPTYLAAAASTPASVWLPVPRGAGPGAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
[GENETYX-MAC:
Multiple Alignment]
OsFER2
1 -MLPPRVAPSSLAAAAAAAPTYLAAAASTPASVWLPVPRGAGAVAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
Date
: 2013.03.08
SoyFERH1
1 MALAPSKVSTFSGFSPKPSVGGAQKNPTCSVSLSFLNEKLGSRNLRVCAST---VPLTGVIFEPFEEVKKSELAVPTAPQVSLARQNYAD
SoyFERH2
1 MALSCSKVLSFY-LSPVVGGGDVPKKLTFSSFLGLSKGVGGSRSSRVCAASNAPAPLAGVIFEPFQELKKDYLAVPIAHNVXLARQNYAD
[GENETYX-MAC:
Multiple Alignment]
SoyFERH3
1 --MLLRTASSFSLLKANADHILPLPNSSSSGIIRYSQSLGKNLVPCATKD-TNNRPLTGVVFEPFEEVKKELDLVPTVPQASLARQKYTD
OsFER1
1 -----MLPPRVAPAAAAAAPTYLAAAASTPASVWLPVPRGAGPGAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
Date
: 2013.03.08
SoyFERH4
1 -----------MLLRTAAASASSLSLFSPNAEPPRSVP-ARGLVVRAAKGSTNHRALTGVIFEPFEEVKKELDLVPTVPQASLARQKYVD
OsFER2
1 -MLPPRVAPSSLAAAAAAAPTYLAAAASTPASVWLPVPRGAGAVAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
Consensus
1 --------------------------------------------------------LXGVXFZPFZEXKXXXXXVPXXXXXXLARQXXXD
SoyFERH1
1 MALAPSKVSTFSGFSPKPSVGGAQKNPTCSVSLSFLNEKLGSRNLRVCAST---VPLTGVIFEPFEEVKKSELAVPTAPQVSLARQNYAD
SoyFERH2
1 MALSCSKVLSFY-LSPVVGGGDVPKKLTFSSFLGLSKGVGGSRSSRVCAASNAPAPLAGVIFEPFQELKKDYLAVPIAHNVXLARQNYAD
OsFER1
1 -----MLPPRVAPAAAAAAPTYLAAAASTPASVWLPVPRGAGPGAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
86
ECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYGY
SoyFERH3
1 --MLLRTASSFSLLKANADHILPLPNSSSSGIIRYSQSLGKNLVPCATKD-TNNRPLTGVVFEPFEEVKKELDLVPTVPQASLARQKYTD
OsFER2
1 -MLPPRVAPSSLAAAAAAAPTYLAAAASTPASVWLPVPRGAGAVAVCRAAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVD
90
ECEAAISEQINVEFNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLMKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYAM
SoyFERH4
1 -----------MLLRTAAASASSLSLFSPNAEPPRSVP-ARGLVVRAAKGSTNHRALTGVIFEPFEEVKKELDLVPTVPQASLARQKYVD
SoyFERH1
1 MALAPSKVSTFSGFSPKPSVGGAQKNPTCSVSLSFLNEKLGSRNLRVCAST---VPLTGVIFEPFEEVKKSELAVPTAPQVSLARQNYAD
88
ECESAINEQINVEYNASYVYHSLFAYFDRDNVALKGFAKFFKESSEEEREHAEKLMKYQNTRGGRVVLHPIKNAPSEFEHVEKGDALYAM
Consensus
1 --------------------------------------------------------LXGVXFZPFZEXKXXXXXVPXXXXXXLARQXXXD
SoyFERH2
1 MALSCSKVLSFY-LSPVVGGGDVPKKLTFSSFLGLSKGVGGSRSSRVCAASNAPAPLAGVIFEPFQELKKDYLAVPIAHNVXLARQNYAD
90
DSESAINEQINVEYNVSYVYHALFAYFDRDNIALKGLAKFFKESSEEEREHAEQLIKYQNIRGGRVVLHPITSPPSEFEHSEKGDALYAM
SoyFERH3
1 --MLLRTASSFSLLKANADHILPLPNSSSSGIIRYSQSLGKNLVPCATKD-TNNRPLTGVVFEPFEEVKKELDLVPTVPQASLARQKYTD
88
DCEATINEQINVEYNVSYVYHAMFAYFDRDNVALKGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSEFDHEEKGDALYAM
OsFER1
86 ECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYGY
SoyFERH4
1 -----------MLLRTAAASASSLSLFSPNAEPPRSVP-ARGLVVRAAKGSTNHRALTGVIFEPFEEVKKELDLVPTVPQASLARQKYVD
79
ESESAVNEQINVEYNVSYVYHAMFAYFDRDNVALRGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSDFDHADKGDALHAM
OsFER2
90 ECEAAISEQINVEFNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLMKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYAM
Consensus
1 --------------------------------------------------------LXGVXFZPFZEXKXXXXXVPXXXXXXLARQXXXD
35
XXEXXXXEQINVEXNXSYXYHXXFAYFDRDNXALXGXAKFFKESSXEERXHAEXLXXYQNXRGGXVXLXXIXXXXXXFXHXXKGDALXXX
SoyFERH1
88 ECESAINEQINVEYNASYVYHSLFAYFDRDNVALKGFAKFFKESSEEEREHAEKLMKYQNTRGGRVVLHPIKNAPSEFEHVEKGDALYAM
SoyFERH2
90 DSESAINEQINVEYNVSYVYHALFAYFDRDNIALKGLAKFFKESSEEEREHAEQLIKYQNIRGGRVVLHPITSPPSEFEHSEKGDALYAM
OsFER1
86 ECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYGY
176
FVL-----------------------------------------------------------------------------------SoyFERH3
88 DCEATINEQINVEYNVSYVYHAMFAYFDRDNVALKGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSEFDHEEKGDALYAM
OsFER2
90 ECEAAISEQINVEFNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLMKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYAM
180
ELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVKNLLPTWVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA--SoyFERH4
79 ESESAVNEQINVEYNVSYVYHAMFAYFDRDNVALRGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSDFDHADKGDALHAM
SoyFERH1
88
178 ECESAINEQINVEYNASYVYHSLFAYFDRDNVALKGFAKFFKESSEEEREHAEKLMKYQNTRGGRVVLHPIKNAPSEFEHVEKGDALYAM
ELALSLEKLVNEKLLNVHSVADRNNDPQMADFIESEFLSEQV--------ESIKKISEYVAQLRRVGKGHGVWHFDQRLLD-----Consensus
35 XXEXXXXEQINVEXNXSYXYHXXFAYFDRDNXALXGXAKFFKESSXEERXHAEXLXXYQNXRGGXVXLXXIXXXXXXFXHXXKGDALXXX
SoyFERH2
90 DSESAINEQINVEYNVSYVYHALFAYFDRDNIALKGLAKFFKESSEEEREHAEQLIKYQNIRGGRVVLHPITSPPSEFEHSEKGDALYAM
180
ELALSLEKLTNEKLLHVHSVAERNNDPQXADFIESEFLYEQV--------KSIKKIAEYVAQLRLVGKGHGVWHFDQKLLHDEDHVSoyFERH3
88 DCEATINEQINVEYNVSYVYHAMFAYFDRDNVALKGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSEFDHEEKGDALYAM
178
ELALSLEKLTNEKLLNLHSVASKNNDVQLADFIESEFLGEQV--------EAIKKISEYVAQLRRVGKGHGVWHFDQMLLHEEGVAA
OsFER1
176 FVL-----------------------------------------------------------------------------------SoyFERH4
79 ESESAVNEQINVEYNVSYVYHAMFAYFDRDNVALRGLAKFFKESSEEEREHAEKLMEYQNKRGGKVKLQSIVMPLSDFDHADKGDALHAM
169
ELALSLEKLTNEKLLNLHSVATKNGDVQLADFVETEYLGEQV--------EAIKRISEYVAQLRRVGKGHGVWHFDQMLLHEGGDAA
OsFER2
180 ELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVKNLLPTWVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA--Consensus
35 XXEXXXXEQINVEXNXSYXYHXXFAYFDRDNXALXGXAKFFKESSXEERXHAEXLXXYQNXRGGXVXLXXIXXXXXXFXHXXKGDALXXX
125
XXXLXLEKLXNEKLXXXHSVAXXXXDXQXXDFXEXEXLXEQVKNLLPTWVXXIKXIXEYVAQLRXVGKGHGVWHFDQXLLXXXXXXA
SoyFERH1
178 ELALSLEKLVNEKLLNVHSVADRNNDPQMADFIESEFLSEQV--------ESIKKISEYVAQLRRVGKGHGVWHFDQRLLD-----SoyFERH2
180 ELALSLEKLTNEKLLHVHSVAERNNDPQXADFIESEFLYEQV--------KSIKKIAEYVAQLRLVGKGHGVWHFDQKLLHDEDHVOsFER1
176 FVL-----------------------------------------------------------------------------------SoyFERH3
178 ELALSLEKLTNEKLLNLHSVASKNNDVQLADFIESEFLGEQV--------EAIKKISEYVAQLRRVGKGHGVWHFDQMLLHEEGVAA
OsFER2
180 ELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVKNLLPTWVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA--SoyFERH4
169 ELALSLEKLTNEKLLNLHSVATKNGDVQLADFVETEYLGEQV--------EAIKRISEYVAQLRRVGKGHGVWHFDQMLLHEGGDAA
SoyFERH1
178 ELALSLEKLVNEKLLNVHSVADRNNDPQMADFIESEFLSEQV--------ESIKKISEYVAQLRRVGKGHGVWHFDQRLLD-----Consensus
125 XXXLXLEKLXNEKLXXXHSVAXXXXDXQXXDFXEXEXLXEQVKNLLPTWVXXIKXIXEYVAQLRXVGKGHGVWHFDQXLLXXXXXXA
SoyFERH2
180 ELALSLEKLTNEKLLHVHSVAERNNDPQXADFIESEFLYEQV--------KSIKKIAEYVAQLRLVGKGHGVWHFDQKLLHDEDHVSoyFERH3
178 ELALSLEKLTNEKLLNLHSVASKNNDVQLADFIESEFLGEQV--------EAIKKISEYVAQLRRVGKGHGVWHFDQMLLHEEGVAA
SoyFERH4
169 ELALSLEKLTNEKLLNLHSVATKNGDVQLADFVETEYLGEQV--------EAIKRISEYVAQLRRVGKGHGVWHFDQMLLHEGGDAA
Consensus
125 XXXLXLEKLXNEKLXXXHSVAXXXXDXQXXDFXEXEXLXEQVKNLLPTWVXXIKXIXEYVAQLRXVGKGHGVWHFDQXLLXXXXXXA
helix
- helix
C- helix
D- helix
F- helix
Supplementary Fig. 5 In silico analysis o f amino acid sequence of ferritin from soybean and rice
85
89
87
89
87
85
78
89
34
87
89
85
175
87
89
179
78
87
177
34
89
179
87
177
175
78
168
179
34
124
177
179
175
178
177
179
263
168
177
250
124
179
257
177
256
178
168
247
263
124
211
250
257
178
256
263
247
250
211
257
256
247
211