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FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: ENEA-Italian National Agency for New Technologies, Energy and Sustainable Economic Development, CASACCIA Research Center, Rome, Italy.

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.

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High purity flow sorting of (AG)12-Cy3 labeled 5B and 3B chromosomes from bread wheat line CSdDt5A.Two sorting regions can be drawn on a DAPI-DNA fluorescence versus a (AG)12-Cy3 FISHIS fluorescence dot plot, which enclose chromosomes 5B and 3B at a purity of 91 and 99%, respectively. Chromosome arms 5AS and 5AL are also bordered to show the high selectivity for chromosome labeling of the probe (AG)12, which do not alter the discrimination of the other remaining chromosomes and chromosome arms, as shown for (GAA)7 labeling (Figure 4). Bar  = 10 µm.
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pone-0057994-g005: High purity flow sorting of (AG)12-Cy3 labeled 5B and 3B chromosomes from bread wheat line CSdDt5A.Two sorting regions can be drawn on a DAPI-DNA fluorescence versus a (AG)12-Cy3 FISHIS fluorescence dot plot, which enclose chromosomes 5B and 3B at a purity of 91 and 99%, respectively. Chromosome arms 5AS and 5AL are also bordered to show the high selectivity for chromosome labeling of the probe (AG)12, which do not alter the discrimination of the other remaining chromosomes and chromosome arms, as shown for (GAA)7 labeling (Figure 4). Bar  = 10 µm.

Mentions: The FISHIS high resolution power can be corroborated by the observation that labeled chromosome 7A from bread and pasta wheat showed the same hybridization pattern as reported previously [10], [22]. In bread wheat, this chromosome exhibits distal (telomeric) bands on both arms (Figure 4) whereas its 7A counterpart in pasta wheat exhibits a distal band only on the long arm (Figure S2). For both pasta and bread wheat, the (AG)12–Cy3 probe revealed two strong hybridization bands on chromosome 5B, and single weak signals on chromosomes 1B, 3B, and 6B [17]. Using this probe, we were able to sort chromosome 5B and 3B to a purity level above 90% (Figure 5).


FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S - PLoS ONE (2013)

High purity flow sorting of (AG)12-Cy3 labeled 5B and 3B chromosomes from bread wheat line CSdDt5A.Two sorting regions can be drawn on a DAPI-DNA fluorescence versus a (AG)12-Cy3 FISHIS fluorescence dot plot, which enclose chromosomes 5B and 3B at a purity of 91 and 99%, respectively. Chromosome arms 5AS and 5AL are also bordered to show the high selectivity for chromosome labeling of the probe (AG)12, which do not alter the discrimination of the other remaining chromosomes and chromosome arms, as shown for (GAA)7 labeling (Figure 4). Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585268&req=5

pone-0057994-g005: High purity flow sorting of (AG)12-Cy3 labeled 5B and 3B chromosomes from bread wheat line CSdDt5A.Two sorting regions can be drawn on a DAPI-DNA fluorescence versus a (AG)12-Cy3 FISHIS fluorescence dot plot, which enclose chromosomes 5B and 3B at a purity of 91 and 99%, respectively. Chromosome arms 5AS and 5AL are also bordered to show the high selectivity for chromosome labeling of the probe (AG)12, which do not alter the discrimination of the other remaining chromosomes and chromosome arms, as shown for (GAA)7 labeling (Figure 4). Bar  = 10 µm.
Mentions: The FISHIS high resolution power can be corroborated by the observation that labeled chromosome 7A from bread and pasta wheat showed the same hybridization pattern as reported previously [10], [22]. In bread wheat, this chromosome exhibits distal (telomeric) bands on both arms (Figure 4) whereas its 7A counterpart in pasta wheat exhibits a distal band only on the long arm (Figure S2). For both pasta and bread wheat, the (AG)12–Cy3 probe revealed two strong hybridization bands on chromosome 5B, and single weak signals on chromosomes 1B, 3B, and 6B [17]. Using this probe, we were able to sort chromosome 5B and 3B to a purity level above 90% (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: ENEA-Italian National Agency for New Technologies, Energy and Sustainable Economic Development, CASACCIA Research Center, Rome, Italy.

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.

Show MeSH
Related in: MedlinePlus