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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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(GAA)7-FITC labeling and FISHIS-based flow-sorting of pasta wheat chromosomes belonging to the homeologous genomes A and B.a) Metaphase chromosomes of pasta wheat labeled by (GAA)7-FITC after ND-FISH on a microscope slide; b) sorting of all chromosomes after FISHIS; c and d) flow-based separation of the A- (low labeling intensity) and B-genome chromosome fractions, respectively. Bar  = 10 µm.
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pone-0057994-g003: (GAA)7-FITC labeling and FISHIS-based flow-sorting of pasta wheat chromosomes belonging to the homeologous genomes A and B.a) Metaphase chromosomes of pasta wheat labeled by (GAA)7-FITC after ND-FISH on a microscope slide; b) sorting of all chromosomes after FISHIS; c and d) flow-based separation of the A- (low labeling intensity) and B-genome chromosome fractions, respectively. Bar  = 10 µm.

Mentions: In pasta wheat, the standard mono-parametric flow karyotype based on DAPI staining comprises three major peaks, only one of them containing a single-type chromosome, that is 3B [22]. The FISHIS based karyotype combining DAPI and (GAA)7-FITC fluorescence resolved several chromosome clusters (Figure 2). Interestingly, differences in the abundance of the GAA motif between the two wheat genomes were clear enough (Figure S2) to provide easy separation of the A- and B-genome chromosomes (Figure 3). When chromosomes present in each cluster were assessed for overall morphology and (GAA)7 hybridization patterns, it became clear that the A-genome chromosomes, which are less intensely labeled than those in the B-genome, were allocated in the regions of lower (GAA)7-FITC fluorescence intensity in both mono- and bi-parametric flow karyograms (Figure S4;Figure 2; regions R1, R2, R3). On the other hand, the B-genome chromosomes, all of which show strong and complex (GAA)7 hybridization patterns, were found in regions corresponding to higher levels of fluorescence within the karyograms (Figure S4;Figure 2; regions R4, R5). At the level of individual chromosomes, FISHIS based on (GAA)7-FITC labeling pattern permitted flow sorting of chromosome 1A to a purity of >92%, chromosome 6A to >93% purity, chromosome 2B to >93% purity, and chromosome 3B to 99% purity (Figure 2).


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)

(GAA)7-FITC labeling and FISHIS-based flow-sorting of pasta wheat chromosomes belonging to the homeologous genomes A and B.a) Metaphase chromosomes of pasta wheat labeled by (GAA)7-FITC after ND-FISH on a microscope slide; b) sorting of all chromosomes after FISHIS; c and d) flow-based separation of the A- (low labeling intensity) and B-genome chromosome fractions, respectively. Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057994-g003: (GAA)7-FITC labeling and FISHIS-based flow-sorting of pasta wheat chromosomes belonging to the homeologous genomes A and B.a) Metaphase chromosomes of pasta wheat labeled by (GAA)7-FITC after ND-FISH on a microscope slide; b) sorting of all chromosomes after FISHIS; c and d) flow-based separation of the A- (low labeling intensity) and B-genome chromosome fractions, respectively. Bar  = 10 µm.
Mentions: In pasta wheat, the standard mono-parametric flow karyotype based on DAPI staining comprises three major peaks, only one of them containing a single-type chromosome, that is 3B [22]. The FISHIS based karyotype combining DAPI and (GAA)7-FITC fluorescence resolved several chromosome clusters (Figure 2). Interestingly, differences in the abundance of the GAA motif between the two wheat genomes were clear enough (Figure S2) to provide easy separation of the A- and B-genome chromosomes (Figure 3). When chromosomes present in each cluster were assessed for overall morphology and (GAA)7 hybridization patterns, it became clear that the A-genome chromosomes, which are less intensely labeled than those in the B-genome, were allocated in the regions of lower (GAA)7-FITC fluorescence intensity in both mono- and bi-parametric flow karyograms (Figure S4;Figure 2; regions R1, R2, R3). On the other hand, the B-genome chromosomes, all of which show strong and complex (GAA)7 hybridization patterns, were found in regions corresponding to higher levels of fluorescence within the karyograms (Figure S4;Figure 2; regions R4, R5). At the level of individual chromosomes, FISHIS based on (GAA)7-FITC labeling pattern permitted flow sorting of chromosome 1A to a purity of >92%, chromosome 6A to >93% purity, chromosome 2B to >93% purity, and chromosome 3B to 99% purity (Figure 2).

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