Supporting Information
Statistical analysis
Two gene model
Multivariate correlation was examined for all pairs of genes in each construct. Expression variability of each
gene is plotted by construct and by event respectively. The following linear mixed model was used to examine the
effects of construct and generation on gene expression:
Log (RNA expression) = construct + generation + generation*construct+ event [: construct] + generation* event [:
construct]
Where construct, generation, and generation*construct are fixed effects, and event [: construct] and generation* event
[: construct] are modeled as random effects. Note that the gene expression levels were log transformed due to the
observation of heterogeneous variances and deviation from normality.
Four gene model
Multivariate correlation was examined for all pairs of genes in each construct. Expression variability of each
gene is plotted by construct and by event respectively. Linear mixed models were applied to log transformed RNA and
protein levels since the original data show non-normality and heterogeneous variances. For AAD1, Cry34Ab1 and
Cry35Ab1, the following model was used:
Log (Expression) = construct + generation+ generation*construct+ event [: construct] + generation* event [: construct]
Where construct, generation, and generation*construct are fixed effects, and event [: construct] and generation* event
[: construct] are modeled as random effects.
For YFP and PAT, the following model was used since only one construct 108717 contains these two genes:
Log (YFP_protein) = generation + event [: construct] + generation* event [: construct]
Where generation is fixed effect, and event [: construct] and generation* event [: construct] are modeled as random
effects.
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Kumar et al. Transgene stacking in plants
Supplementary Table 1: Correlation between gene pairs for transcripts obtained from construct 108717
YFP_RNA
AAD1_RNA
Cry34_RNA
Cry35_RNA
YFP_RNA
1.0000
0.7845
0.7240
0.6193
AAD1_RNA
0.7845
1.0000
0.5958
0.5336
Cry34_RNA
0.7240
0.5958
1.0000
0.9164
Cry35_RNA
0.6193
0.5336
0.9164
1.0000
Supplementary Table 2: Correlation between gene pairs for proteins obtained from construct 108717
YFP_Protein
AAD1_Protein
Cry34_Protein
Cry35_Protein
YFP_Protein
1.0000
0.7389
0.8644
0.8062
AAD1_Protein
0.7389
1.0000
0.5476
0.5247
Cry34_Protein
0.8644
0.5476
1.0000
0.9671
Cry35_Protein
0.8062
0.5247
0.9671
1.0000
Supplementary Table 3: Correlation between gene pairs for transcripts obtained from construct 105818
AAD1_RNA
Cry34_RNA
Cry35_RNA
AAD1_RNA
1.0000
0.8397
-0.5955
Cry34_RNA
0.8397
1.0000
-0.7514
Cry35_RNA
-0.5955
-0.7514
1.0000
Supplementary Table 4: Correlation between gene pairs for proteins obtained from construct 105818
AAD1_Protein
Cry34_Protein
Cry35_Protein
AAD1_Protein
1.0000
0.3304
-0.0787
Cry34_Protein
0.3304
1.0000
-0.6728
Cry35_Protein
-0.0787
-0.6728
1.0000
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Kumar et al. Transgene stacking in plants
108706
101556
Control
YFP
YFP
YFP
GUS
GUS
GUS
Supplementary Figure 1: Transient YFP expression in maize immature embryos. Representative embryo
showing transient YFP and GUS expression obtained from ZMUbi bidirectional promoter construct 108706
compared to the unidirectional ZMUbi promoter driving yfp construct 101556. The non-transformed control
embryo is also shown. The assays were done 3 days after Agrobacterium co-cultivation.
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Kumar et al. Transgene stacking in plants
108707
101556
108716
Negative control
Supplementary Figure 2: Transient YFP expression in maize immature embryos. Representative embryo
showing transient YFP and GUS expression obtained from ZMUbi bidirectional promoter construct 108707
compared to 108716 construct lacking minP and the unidirectional ZMUbi promoter driving yfp construct
101556. No YFP expression was detected from the construct 108716. The non-transformed control embryo is
also shown. The assays were done 3 days after Agrobacterium co-cultivation.
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Kumar et al. Transgene stacking in plants
Supplementary Figure 3: Plasmids Maps
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Kumar et al. Transgene stacking in plants
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Kumar et al. Transgene stacking in plants
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Kumar et al. Transgene stacking in plants
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Kumar et al. Transgene stacking in plants