Supplementary procedure:
DNA analysis
DNA was isolated by the Cetyl Triethyl Ammonium Bromide method from pooled
seedlings or from leaves and floral whorls of individual plants. DNA was analyzed by
polymerase chain reaction (PCR) and Southern hybridization. PCR was carried out to 39
cycles using a MyCycler® thermocycler, and the resulting PCR products were run on
agarose gels and visualized under UV. Primers used for detecting SJN03 transformation
(see Fig. 4) were the following: a, 5’-AGTTTGAGATCAAGACATCTG-3’; b, 5’GTGCTGAACAATGCTGTGAC-3’; and c, 5’-AAACGACAATCTGATCCAAG-3’.
Southern blots were prepared by blotting with 0.8% agarose gels on Amersham Hybond
N+® membranes.
Southern blots were then probed with P32 labeled DNA probes,
washed and visualized using phosphorimager.
Supplementary Figure Legends:
Figure 1: Analysis of copy number and direct repeat structures in 68LF lines. (a) BamHI
(B) map of a direct repeat locus generated by 68LF. A diagnostic 3 kb band is
expected from a direct RB::LB repeat structure, while each copy would produce a 5.8
kb internal band. Location of DNA probes, ‘a’ and ‘b’ is indicated by solid lines, (bc) Southern hybridization of BamHI digested genomic DNA of 68LF lines with probe
‘a’ and ‘b’. M, molecular size markers (in kb); N, non-silenced lines.
Figure 2: Analysis of indirect repeat structures in 68LF lines. (a-b) HindIII (H) map of
indirect repeat locus of 68LF (LB::LB and RB::RB). DNA probe fragments (a, b) are
indicated by solid lines. (c-d) Southern blot of HindIII digested genomic DNA of
68LF lines hybridized with probe ‘a’ and ‘b’. M, molecular size markers (in kb); N,
non-silenced lines.
Figure 3: Detection of SJN03 integration into T0 seedlings. (a). Map of SJN03, a
promoterless and marker-free construct. To determine the integration of SJN03 TDNA, DNA from bulked T0 seedlings was subjected to PCR with PHYA-specific
forward primers, a and b, and a T-DNA RB reverse primer, c. Expected product sizes
are listed for each primer set. (b). PCR on DNA isolated from a pool of SJN03 T0
seedlings using a-c and b-c primer pairs. WT and No DNA lanes are included as
negative controls, and the 68LF-35 line is used as a positive control. The 3’ termini
of 68LF and SJN03 are identical (see Fig 3). Bands of the correct size were produced
in SJN03 T0 DNA with each primer set, indicating successful SJN03 integration.
Figure 4: Analysis of transgene copy number in SJN02 lines. (a) EcoRV (RV) map of the
SJN02 construct. Flanking fragments are indicated by dashed arrows.
Probe
fragments (a and b) are shown by solid lines. (c-d) Southern blot of EcoRV digested
genomic DNA of SJN02 lines hybridized with probe ‘a’ or ‘b’. M, molecular size
markers (in kb); N, non-silenced lines; SC, single-copy pattern
Figure 5: Analysis of transgene copy number in SJN04 lines (1 to 10). (a) EcoRV (RV)
map of the SJN04 construct. Indirect repeat (5.4 kb) and internal (3.8 kb) fragments
are shown. Probe fragments (a and b) are shown by solid lines. (b-c) Southern blot of
EcoRV digested genomic DNA of SJN04 lines hybridized with probe ‘a’ or ‘b’. M,
molecular size markers (in kb); N, non-silenced lines; SC, single-copy patterns.