Figure 4

FISH of Chinese chestnut CC4 with rDNA probes. Chinese chestnut CC4 pro-metaphase chromosome spread hybridized with 35S (green) and 5S (red) rDNA probes; (a) SAT-1 and SAT-2 are different sizes (double arrowheads) with proximal 35S co-localized with the second 5S rDNA site (red), mn-35S site (arrows) and 5S site (arrowheads), one of the mother chromosomes encircled with white dotted line (middle-left); (b) same cell as in ‘a’ under red and blue filters shows two pairs of 5S sites (arrows and double arrowheads), the satellites with the second 5S site (double arrowheads) appear detached from the mother chromosomes, DAPI stained image of the satellites shown in insert (middle-slight right); (c) another cell of an early pro-metaphase spread with 35S signals. The satellites are marked by double arrowheads and the mother chromosomes are encircled (white dotted lines), the mn-35S site marked by arrows; (d) same cell as in ‘c’ with reduced DAPI (image processed further) to clearly demonstrate the 35S FISH signals, which are seen only in the weakly stained region of the satellites (brace, see enlarged DAPI image in ‘f4’ and ‘f8’, and clear separation between the two regions marked by bent arrows); (e) same cell as in ‘c' with reduced DAPI that clearly demonstrated the distal region of the satellites (arrowheads) is highly heterochromatic as it fluoresced intensely under blue filter; (f) enlarged images of SAT-1 and SAT-2 under different filters clearly demonstrating the proximal region of each satellite may be euchromatic as this region is integrated with the 35S rRNA gene (35S green signals), SAT-1 under green and blue filters (f1), image with reduced DAPI (f2), image with reduced green (f3) and DAPI image (f4) clearly shows differential DAPI intensities, the proximal region stained weakly (brace) and distal region stained intensely (highly heterochromatic), arrows path shows clear separation line between these two chromatin regions, for f5–f8 (SAT-2) the description is same as in f1–f4, respectively; (g) diagrammatic illustration of the rDNA bearing chromosomes (abbreviations are same as in Fig. 1f). Bars = 5 µm (b,c), and = 0.5 µm.