Figure 3

FISH of Chinese chestnut CC3 with rDNA probes. Pro-metaphase and early metaphase chromosome spreads of Chinese chestnut CC3 hybridized with 35S (green signals) and 5S (red signals) probes; a) two satellites [SAT-1 (white dotted lined box, inside the large rectangular box), SAT-2 (double arrowheads in the large rectangular box)] associated with the mj-35S signals, two mn-35S signals (arrows) and two 5S signals (arrowheads), an enlarged image of the SAT-1 under different filter shown in inserts (top, 1 to 5) to demonstrate the 35S and the second 5S positions (localizations) in the satellite; image under red–green–blue (UV) filters (a1), green–blue filters (a2), red-green filters (a3), red-blue filters (a4) and blue filter (a5); (b) an enlarged image of the mj-35S bearing mother chromosomes and the satellites (large rectangular box in ‘a’) are seen to be connected (white dotted lines with arrowheads); (c) when FISH signals are removed (image process) leaving only the DAPI stain, both satellites can be seen clearly [an enlarged image of the satellites, SAT-1 (white dotted lined box) and SAT-2 (arrowhead)]; (d) an advanced pro-metaphase (or early metaphase) spread with 35S rDNA signals (green), SAT-1 chromosome is seen intact [arrowhead (top-left), SAT-1 with reduced green signal (d1) and DAPI image (d2)], SAT-2 is covered by green signal (double arrowhead), which is seen to be connected with its mother chromosome [(white dotted lines with arrowheads), SAT-2 with reduced green signal (d3) and DAPI image (d4)]; (e) an early metaphase, both SAT-1 (arrowhead) and SAT-2 (double arrowhead) seen to be clearly attached with respective mother chromosomes (each encircled with white dotted lines); SAT-1 with reduced green signal (e1, image processed further), DAPI image of SAT-1 (e2), SAT-2 with reduced green signal (e3, image processed further) and DAPI image (e4); (f) diagrammatic illustrations of the rDNA bearing chromosomes (abbreviations are same as in Fig. 1f). Bars = 5 µm.