A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants

Abstract

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4',6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.

Investigation of Chromosomes in Varieties and Translocation Lines of Pea Pisum sativum L. by FISH, Ag-NOR, and Differential DAPI Staining

“…The total number of the DAPI bands per haploid pea genome is 344. For comparison: 185 DAPI bands per haploid genome are observed on metaphase barley chromosomes after deconvolution, though the genome size in barley is larger than in pea [23]. Thus, the use of intercalator 9-aminoachridine significantly increases resolution of differential banding of pea chromosomes, as was earlier shown for other organisms [21,[24][25][26].…”

Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

“…However, it is difficult to produce identifiable bands in plant chromosomes stained by various dyes due to the coarseness in the pattern of the bands. The development of computer image processing and analysis techniques, especially the technique of 3D reconstruction and deconvolution, could improve banding resolution and offer opportunities to obtain clear bands [33]. In maize meiotic pachytene chromosomes, 3D imaging data provided a complete view of various histone methylation distributions [12].…”

The Arabidopsis LHP1 protein is a component of euchromatin

“…A ladder of polynucleosomes is observed after 30 s on the lhp1 nuclei (1: mononucleosome, 2: dinucleosomes...). Ld: 100 bp DNA Ladder, a: 600-bp fragment known from several studies (Moscone et al 1996;Ali et al 2000;Liu et al 2004). However, such large AT-rich segments are not present in the A. thaliana genome.…”