Figure 3

Chromosomal localization of Lyc EPRVs. Double target fluorescent in situ hybridization was carried out on root tip metaphases and male meiotic pachytene cells of S.lycopersicum (A-F) and S.habrochaites (G-I). Biotin labelled pooled probes of LycEPRVs from S.lycopersicum (LycEPRV-Sl, A-F) and S.habrochaites (LycEPRV-Sh, G-I), respectively, that cover most of LycEPRV sequence (for clone combinations see Table 2) were detected by red Alexa-594 fluorescence and hybridized together with digoxigenin labelled repeated DNA probes detected by green FITC fluorescence. Chromosomes were counterstained with DAPI (blue fluorescence). A-C) Metaphase chromosomes of S.lycopersicum (2n = 24). LycEPRV-Sl sequences (red in B and magenta in the overlay with blue DAPI staining in A) are located at the centromeres of most chromosomes with variable intensity, but are absent from the NOR region (green rDNA probe in A) and reduced on four chromosomes (arrows in B). In C the LycEPRVs are shown to co-localize with the retroelement sequence U30 from S.lycopersicum (green) that shows dispersed signals on all chromosomes. D-F) Pachytene chromosomes of S.lycopersicum are much more extended than metaphase chromosomes and show differentiation with DAPI into strongly stained heterochromatin and weakly stained euchromatin (D). The red LycEPRV signal is almost exclusively seen in the pericentromeric heterochromatic regions and intercalary chromocentre (arrowheads in D and E), but not at the NOR region (green in E, F; DAPI is shown as grey image with the probe signal falsely coloured red and green, respectively). G-I) Metaphase chromosomes of S.habrochaites (2n = 24). LycEPRV-Sh sequences (red in H, magenta in the overlay with blue DAPI staining in G, I) are located near the centromeres of most chromosomes showing stronger signal in some. No signal is visible in the NOR regions (green rDNA probe in G, arrow heads in I). Bar 10 μm.