Supplemental Method:

Real-time quantitative PCR: Real Time quantitative Polymerase Chain Reaction (RT-qPCR) was carried out on Mx3000P (Stratagene) thermocycler. RT-qPCR on total RNA isolated using RNAesy kit (Invitrogen Inc) was carried out using SuperScript III Platinum SYBR Green One-Step Kit (Invitrogen Inc.). Reactions were prepared according to the protocol provided in the kit and optimized for 0.5 ng of total RNA. Cycle threshold (CT) value obtained in this assay is used as a direct measure of transcript dosage. An inverse correlation between CT value and transcript dosage is expected. Primer sequences used in this assay are given below:

GFP Forward : 5’-AGGACGACGGCAACTACAAG-3’

GFP Reverse : 5’-GATGGGGGTGTTCTGCTG-3’

GUS Forward : 5’-TGATCAGCGTTGGTGGGAAAGC-3’

GUS Reverse : 5’- GTGGTGTAGAGCATTACGCTGC-3’

PDS Forward : 5’-GCAGAGGAATGGGTTGGAC-3’

PDS Reverse : 5’-AGAGGTCGGCAAGGTTCAC-3’

Supplemental Data:

Supplemental Table 1: RT-qPCR on GUS lines

GUS
line / Copy # / CT Value1
GUS / PDS3
1-22
1-4
1-5
1-6
1-7
1-8
1-10
1-11
1-12
2-1
2-2
2-5
2-6
2-7
2-8
2-9
3-1
3-4
T5 / 1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
control / >36
32.34
32.03
32.50
32.09
32.39
31.98
32.27
31.99
31.73
ND
ND
31.82
31.50
31.90
ND
30.32
27.43
>35 / ND
25.90
26.06
25.70
24.69
25.86
25.43
26.87
24.53
25.80
ND
ND
24.77
26.05
26.13
ND
24.87
25.67
24.44

1Cycle threshold; 2Silenced; ND, not determined;

3Phytoene desaturase (internal control)

Supplemental Figure 1: Correlation of GUS gene copy number with GUS transcript levels. CT values for amplification of GUS and PDS transcripts in GUS-expressing lines, as shown in Supplemental Table 1, are plotted. PDS serves as an internal control.

Supplemental Table 2: RT-qPCR on GFP lines

GFP
line / Copy # / CT values1
GFP / PDS2
177
178
179
180
181
182
107
154
157
160
162
T5 / 1
1
1
2
2
2
3
3
3
3
3
control / 33.53
32.53
32.14
30.22
30.32
29.81
29.17
29.00
29.28
27.90
27.58
>37 / 25.49
24.87
25.23
25.55
25.20
25.70
25.15
25.73
26.30
25.67
26.81
25.12

1Cycle threshold; 2Phytoene desaturase

(internal control)

Supplemental Figure 2: Correlation of GFP gene copy number with GFP transcript levels. CT values for amplification of GFP and PDS transcripts in GFP lines, as shown in Supplemental Table 2, are plotted. PDS serves as an internal control.

3