Abstract
Background: Supernumerary chromosomes (B) comprise optional complement to basic (A) chromosome set. The presence of B-chromosomes may significantly reduce plant vigor and fertility. Potentially active genes constitute only small fraction of DNA of these chromosomes indicating that these effects are mediated by epigenetic mechanisms. One example is down-regulation of rDNA genes and condensation of their respective chromatin regions (demonstrated in squashed preparations using 2D microscopy). It may be postulated that the presence of B chromosomes leads to more extensive changes of local chromatin structure. Verification of hypothesis requires studying 3D spatial architecture of intact nuclei in tissue. Results: An image processing algorithm was developed and applied for isolation (from the confocal datasets) of regions corresponding to single nuclei. The nuclei were segmented using iterative global thresholding followed by growing and merging of regions belonging to different nuclei. The result of segmentation was verified by a human observer. Chromatin architecture was characterized quantitatively using global fluorescence intensity distribution measures (mean, variance) and local intensity distribution parameters (haraclick features, wavelet energy, run- length features). The sets of parameters corresponding to populations of nuclei with different number of B-chromo- somes were subjected to discriminate analysis. The distinct parameters were then correlated with depth in tissue at which a given nucleus was positioned. Conclusions: Combination of light microscopy with dedicated image processing and analysis framework made it possible to study chromatin architecture in nuclei containing various number of B chromosomes. These data indicate that alterations of 3D chromatin distribution occur globally in the interphase nuclei in the presence of Bs. The changes occur at the spatial scale comparable with the resolution limit of light microscopy and at larger distances.
Highlights
Supernumerary chromosomes (B), present in a number of plant and animal species [1], are facultative complement of basic chromosome set (A)
The B chromosomes are mostly smaller than their counterparts of the A set and their numbers may vary between different organisms in one species or different cells in one organism [2,3] These chromosomes have high content of repetitive DNA sequences and may be abundant in constitutive heterochromatin [3,4,5]
The parameters characterizing local fluorescence intensity distribution were subjected to discriminant analysis to isolate the set of features which were distinct for nuclei containing different numbers of B chromosomes
Summary
Supernumerary chromosomes (B), present in a number of plant and animal species [1], are facultative complement of basic chromosome set (A). Active genes constitute only small fraction of DNA of these chromosomes indicating that these effects are mediated by epigenetic mechanisms. One example is down-regulation of rDNA genes and condensation of their respective chromatin regions (demonstrated in squashed preparations using 2D microscopy). Conclusions: Combination of light microscopy with dedicated image processing and analysis framework made it possible to study chromatin architecture in nuclei containing various number of B chromosomes. These data indicate that alterations of 3D chromatin distribution occur globally in the interphase nuclei in the presence of Bs. The changes occur at the spatial scale comparable with the resolution limit of light microscopy and at larger distances
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