A novel FISH technique for labeling the chromosomes of dinoflagellates in suspension

Dinoflagellates possess some of the largest known genomes. However, the study of their chromosomes is complicated by their similar size and their inability to be distinguished by traditional banding techniques. Dinoflagellate chromosomes lack nucleosomes and are present in a liquid crystalline state. In addition, approaches such as fluorescent in situ hybridization (FISH) are problematic because chromosomes are difficult to isolate from the nuclear membrane, which in dinoflagellates remains intact, also during mitosis. Here we describe a novel, reliable and effective technique to study dinoflagellate chromosomes by physical mapping of repetitive DNA sequences in chromosomes in suspension (FISH-IS), rather than on a microscope slide. A suspension of non-fixed chromosomes was achieved by lysing the cells and destabilizing the nuclear envelope. This treatment resulted in the release of the permanently condensed chromosomes in a high-quality chromosomal suspension. Nevertheless, slide preparations of the chromosomes were not suitable for conventional FISH because the nuclear integrity and chromosomal morphology was destroyed. Our newly developed, simple and efficient FISH-IS technique employs fluorescently labeled, synthetic short sequence repeats that are hybridized with suspended, acetic-acid-pretreated chromosomes for 1 h at room temperature. The method can be successfully used to discriminate single chromosomes or specific chromosomal regions, depending on the specificity of the repeat sequences used as probes. The combination of FISH-IS and flow sorting will improve genomic studies of dinoflagellates, overcoming the difficulties posed by their huge genomes, including long stretches of non-coding sequences in multiple copies and the presence of high-copy-number tandem gene arrays.