Figure 1.
Diagram showing the GM-specific target amplification for each GM event.
Expected amplicons for each primer set are shown below the construct diagram, while the amplicons for the enhanced 35S promoter and the NF3R are displayed above.
Figure 2.
PCR analysis of the monitoring samples.
Panel A reveals three GM events (lanes 1-3) based on amplification of the 35S promoter- and nos-specific bands. Asterisks and triangles indicate unexpected ~400 bp and ~800 bp bands, respectively. Lane N: negative control (no DNA template), M: 1kb+ DNA ladder, CRM: certified reference material with MON810 and MON863 DNA (Sigma Cat. No. ERMBF417D), 1-3: Yeonan Wharf road samples, 4-7 and 11: Wonju samples, 8-10: Incheon field samples, 12: Yeoju sample, 13: Namyangju sample, and 14: Busan sample. Zein was used as a positive control for maize. B: Comparison of the singleplex and quadruplex PCR results of GM sample 3. The absence of the ~800 bp band in reactions 7 and 8 indicates that the 35S and NOS primers are responsible for its amplification. Asterisks and triangles indicate unexpected ~400 bp and ~800 bp bands, respectively.
Figure 3.
PCR and sequence analyses of the ~800 bp band.
Panel A: Amplification diagram of the different combinations of 35S and NOS primers to investigate the primer pair responsible for amplification of the ~800 band and their physical orientation. B: PCR results of primer combinations from panel A. Lane 1: NOSF and 35SF (NF3F), 2: NOSR and 35SR (NR3R), 3: NOSF and 35SR (NF3R), and 4: NOSR and 35SF (NR3F). Only the NF3R combination produced the band, indicating the upstream location of the nos segment to the 35S. C: Confirmation of the NK603 event-specific cassette in GM sample 3. Among the different GM maize events, NK603 contains adjacent nos and 35S loci. The PCR results showed that the NF3R band corresponds to the NK603 event. Lane 1: MON810, 2: NK603-specific, 3: NF3R. These results also demonstrate the stacked event of GM sample 3. D: The consensus sequence of the NF3R band (766 bp, blue arrow). The sequence further confirmed the upstream location of the nos terminator (green arrow) to the 35S (114 bp, purple arrow) and e35S (375 bp, red arrow) promoters. The 35S and NOS primer pairs are shown as gray arrows.
Figure 4.
FISH analysis of the 45S rDNA cluster in ‘Mibaekchal’.
The 5S (green) and 45S (red) rDNA probes showed two signals in both the interphase (A–D) and pro-metaphase chromosomes (E–H). Panels A and E: raw DAPI image, B and F: raw 45S rDNA signal, C and G: overlaid pseudo-colored rDNA signals on raw DAPI images, D and H: merged pseudo-colored images. Yellow arrowheads indicate the knob of a satellite homologue 6. Bar = 5µm.
Figure 5.
FISH analysis of the 45S rDNA cluster in GM sample 3.
In both the interphase (A–E) and metaphase (F–I) spreads, two signals for the 5S rDNA cluster (green) but fragmented patterns for the 45S rDNA cluster (red) were observed. Panel A shows the normal two 45S rDNA signal pattern, while panels B-E show the different numbers of 45S rDNA fragments ranging from three to six. Yellow arrows emphasize the 45S rDNA signals, bar = 5µm. Panels F-I show different metaphase spreads displaying homozygously fragmented 45S rDNA with “beads” (yellow arrows) and “string” (broken lines) patterns. Satellite chromosomes are indicated by the number 6, and asterisks represent the homologue bearing a large knob (green arrowhead), bar = 5µm.
Figure 6.
Diagram showing the “beads-on-a-string” pattern observed in GM sample 3.
The 45S rDNA cluster in the GM sample frequently revealed three “beads” connected by two “strings” (arrows) in metaphase spreads. Panel A: part of a metaphase spread (full image shown in Figure S2, panel D), B: diagram of the image in panel A. Bar = 5µm.
Figure 7.
Genetic pattern of the fragile 45S rDNA locus.
Panel A: FISH analysis of the 45S rDNA locus in F1 metaphase chromosome spreads show one satellited homologue with intact NOR (green arrows), and a fragmented NOR (yellow arrows) from its homologous locus. Bars = 5µm. B: A genetic diagram depicting the inheritance of NOR condensation pattern to the F1 progeny.
Figure 8.
Comparative PCR and FISH analyses between Hi-II and MON810.
Panel A: Multiplex PCR results confirmed the absence or presence of transgene in Hi-II and MON810, respectively. Lane N: negative control (no DNA template), 1: Hi-II, and 2: MON810. B: FISH analysis of the 45S rDNA cluster showed two intact signals in both Hi-II and MON810. Bar = 20µm.