Abstract
The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.
Highlights
Eukaryotic genomes are partitioned into chromosomes, and chromosome numbers vary between species from two to many hundreds [1]
Each chromosome will be characterized by two main features, or parameters: its total DNA content and its FISH labeling fluorescent pattern, and the combination of both parameters will be shown as single dot on a bi-parametric dot plot drawing on the flow cytometer computer screen
The availability of single-type chromosome-specific DNA from major species will facilitate the development of molecular markers from small amounts of flow-sorted chromosomes, and it will enable the construction of highly saturated genetic maps from specific genome regions; it will facilitate the analysis of the haplotype in complex genomes, supporting a comprehensive gene content analysis and gene discovery [51,52,53,54,4]
Summary
Eukaryotic genomes are partitioned into chromosomes, and chromosome numbers vary between species from two to many hundreds [1]. Genomes can be investigated by a number of methodologies, some of which analyze total genomic DNA in bulk, whereas others preserve chromosomal individuality. Plant genomics would necessitate the joining of both methodologies to overcome the huge task of assembling hybrid genomes and to represent the vast diversity existing into the species, and that cannot be shown up by the sequencing of a single individual genome. Even with the powerful third-generation sequencing technologies, this effort would be costly and difficult to apply for large plant polyploid genomes. The improved chromosome approach we propose can be used to reduce the whole-genome complexity, opening an easy access to single chromosomes from several and different species
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