Establishing of 3D-FISH on frozen section and its applying in chromosome territories analysis in Populus trichocarpa

The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biological processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. We focused on the chromosome territory (CT) analysis of P. trichocarpa chromosome 19 in the root tip nuclei. Remarkably, the distance between homologous chromosomes 19 was variable and they were spatially completely separated in 80% of the nuclei. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar.