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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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FISHIS of pasta wheat cv Creso chromosome suspensions.a) Chromosome suspensions hybridized with (GAA)7-FITC; b) flow-sorted chromosomes 3B, 4B and 5B following hybridization with (AG)12-Cy3; c) (GAA)7-FITC and (AAT)7-Cy3 dual labelled chromosomes and nuclei; d) pasta wheat chromosomes and a nucleus after (GAA)7-FITC and (AAC)5-Cy3 dual labeling. Bar  = 10 µm.
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pone-0057994-g001: FISHIS of pasta wheat cv Creso chromosome suspensions.a) Chromosome suspensions hybridized with (GAA)7-FITC; b) flow-sorted chromosomes 3B, 4B and 5B following hybridization with (AG)12-Cy3; c) (GAA)7-FITC and (AAT)7-Cy3 dual labelled chromosomes and nuclei; d) pasta wheat chromosomes and a nucleus after (GAA)7-FITC and (AAC)5-Cy3 dual labeling. Bar  = 10 µm.

Mentions: The optimal pH for DNA denaturation was determined experimentally by the addition of NaOH to a 150 µl aliquot of suspended chromosomes (2×106 chromosomes/ml LB01). The pH measurements were performed with an ISFET micro pH probe (Hatch PHW17-SS) inserted into the sample tube. The extent of DNA denaturation over the pH range 8.0–13.8 was assessed as a time course (0–60 mins) by labeling with 36 µM Acridine Orange (AO) followed by flow cytometry [32] (AO stains single-stranded DNA red and double-stranded DNA green). Flow data were collected as single DNA histograms and dot plots plotting the ratio of ssDNA/dsDNA fluorescence against the dsDNA fluorescence for 104 chromosomes per sample, removing debris and aggregates from the forward scatter signal versus a DAPI fluorescence dot plot. The HPCV (Half Peak Coefficient of Variation: the standard deviation (s) of the fluorescent intensity of a population of chromosomes expressed as a percentage of the mean (m) intensity (CV  =  s/m•100) measured at 50% peak height) was taken as an index for chromosome morphology and DNA integrity [33]. The optimum denaturation treatment was set at pH 13 for 20 mins, followed by the addition of 1M Tris-HCl pH 7.4 and maintaining the suspension on ice for 1 min to return to pH8.0. After denaturation, yield and morphology of chromosomes were evaluated by FCM (Figure S1) and microscope observation (Figure 1).


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)

FISHIS of pasta wheat cv Creso chromosome suspensions.a) Chromosome suspensions hybridized with (GAA)7-FITC; b) flow-sorted chromosomes 3B, 4B and 5B following hybridization with (AG)12-Cy3; c) (GAA)7-FITC and (AAT)7-Cy3 dual labelled chromosomes and nuclei; d) pasta wheat chromosomes and a nucleus after (GAA)7-FITC and (AAC)5-Cy3 dual labeling. Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057994-g001: FISHIS of pasta wheat cv Creso chromosome suspensions.a) Chromosome suspensions hybridized with (GAA)7-FITC; b) flow-sorted chromosomes 3B, 4B and 5B following hybridization with (AG)12-Cy3; c) (GAA)7-FITC and (AAT)7-Cy3 dual labelled chromosomes and nuclei; d) pasta wheat chromosomes and a nucleus after (GAA)7-FITC and (AAC)5-Cy3 dual labeling. Bar  = 10 µm.
Mentions: The optimal pH for DNA denaturation was determined experimentally by the addition of NaOH to a 150 µl aliquot of suspended chromosomes (2×106 chromosomes/ml LB01). The pH measurements were performed with an ISFET micro pH probe (Hatch PHW17-SS) inserted into the sample tube. The extent of DNA denaturation over the pH range 8.0–13.8 was assessed as a time course (0–60 mins) by labeling with 36 µM Acridine Orange (AO) followed by flow cytometry [32] (AO stains single-stranded DNA red and double-stranded DNA green). Flow data were collected as single DNA histograms and dot plots plotting the ratio of ssDNA/dsDNA fluorescence against the dsDNA fluorescence for 104 chromosomes per sample, removing debris and aggregates from the forward scatter signal versus a DAPI fluorescence dot plot. The HPCV (Half Peak Coefficient of Variation: the standard deviation (s) of the fluorescent intensity of a population of chromosomes expressed as a percentage of the mean (m) intensity (CV  =  s/m•100) measured at 50% peak height) was taken as an index for chromosome morphology and DNA integrity [33]. The optimum denaturation treatment was set at pH 13 for 20 mins, followed by the addition of 1M Tris-HCl pH 7.4 and maintaining the suspension on ice for 1 min to return to pH8.0. After denaturation, yield and morphology of chromosomes were evaluated by FCM (Figure S1) and microscope observation (Figure 1).

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