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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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Specific PCR amplification of DNA isolated from flow-sorted FISHIS labeled pasta wheat chromosomes 1A, 6A and 3B.In all Panels: lane M: 50 bp-step ladder; lane WG: whole pasta wheat genomic DNA; lane F-AG: FISHIS labeled flow-sorted whole A-genome chromosomes; lane F-BG: FISHIS labeled flow-sorted whole B-genome chromosomes; lane NF-ALL: whole flow-sorted chromosome complement without FISHIS labeling; lane F-ALL: sorting of the whole FISHIS labeled pasta wheat chromosome complement. Panel a): chromosome 1A amplicons analysis with specific primers from the Xgwm136-1A SSR molecular marker. In lane F-1A, the DNA obtained from 300 FISHIS labeled flow-sorted 1A chromosomes has been PCR amplified showing a band which is only visible where 1A chromosome DNA is present. Panel b): chromosome 6A PCR amplification with primers from the Xgwm169-6A SSR molecular marker. In lane F-6A a specific 6A chromosome band is shown which is absent in all B-genome amplifications. Panel c): chromosome 3B PCR amplification with primers from the Xgwm285-3B SSR molecular marker. In lane F-3B, 300 FISHIS labeled 3B chromosomes have been amplified with a specific probe showing a band present in all lanes where 3B chromosome DNA is present.
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pone-0057994-g007: Specific PCR amplification of DNA isolated from flow-sorted FISHIS labeled pasta wheat chromosomes 1A, 6A and 3B.In all Panels: lane M: 50 bp-step ladder; lane WG: whole pasta wheat genomic DNA; lane F-AG: FISHIS labeled flow-sorted whole A-genome chromosomes; lane F-BG: FISHIS labeled flow-sorted whole B-genome chromosomes; lane NF-ALL: whole flow-sorted chromosome complement without FISHIS labeling; lane F-ALL: sorting of the whole FISHIS labeled pasta wheat chromosome complement. Panel a): chromosome 1A amplicons analysis with specific primers from the Xgwm136-1A SSR molecular marker. In lane F-1A, the DNA obtained from 300 FISHIS labeled flow-sorted 1A chromosomes has been PCR amplified showing a band which is only visible where 1A chromosome DNA is present. Panel b): chromosome 6A PCR amplification with primers from the Xgwm169-6A SSR molecular marker. In lane F-6A a specific 6A chromosome band is shown which is absent in all B-genome amplifications. Panel c): chromosome 3B PCR amplification with primers from the Xgwm285-3B SSR molecular marker. In lane F-3B, 300 FISHIS labeled 3B chromosomes have been amplified with a specific probe showing a band present in all lanes where 3B chromosome DNA is present.

Mentions: Single-type FISHIS chromosome and genome fractions along with the total chromosome content of pasta wheat were flow-sorted to evaluate their suitability as a substrate for direct PCR with chromosome-specific primers. We identified and flow-sorted three pasta wheat chromosome fractions which showed very different labeling intensities (Figure 2), that is, 1A, 6A and 3B (Figure S2 and Figure S5). The products from PCR amplifications with primers derived from the chromosome-specific SSR molecular markers Xgwm136, Xgwm169 and Xgwm285 located on chromosomes 1A, 6A and 3B, respectively, are presented and the expected amplicon bands are shown (Figure 7). Wheat A- and B-genome whole-chromosome complements were flow-sorted after FISHIS labeling, proving the effectiveness of the technique in providing a specific PCR probe-related pattern. No sign of contamination or DNA degradation, due to the presence of other chromosome types or to the FISHIS methodology, respectively, was detected (Figure 7).


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)

Specific PCR amplification of DNA isolated from flow-sorted FISHIS labeled pasta wheat chromosomes 1A, 6A and 3B.In all Panels: lane M: 50 bp-step ladder; lane WG: whole pasta wheat genomic DNA; lane F-AG: FISHIS labeled flow-sorted whole A-genome chromosomes; lane F-BG: FISHIS labeled flow-sorted whole B-genome chromosomes; lane NF-ALL: whole flow-sorted chromosome complement without FISHIS labeling; lane F-ALL: sorting of the whole FISHIS labeled pasta wheat chromosome complement. Panel a): chromosome 1A amplicons analysis with specific primers from the Xgwm136-1A SSR molecular marker. In lane F-1A, the DNA obtained from 300 FISHIS labeled flow-sorted 1A chromosomes has been PCR amplified showing a band which is only visible where 1A chromosome DNA is present. Panel b): chromosome 6A PCR amplification with primers from the Xgwm169-6A SSR molecular marker. In lane F-6A a specific 6A chromosome band is shown which is absent in all B-genome amplifications. Panel c): chromosome 3B PCR amplification with primers from the Xgwm285-3B SSR molecular marker. In lane F-3B, 300 FISHIS labeled 3B chromosomes have been amplified with a specific probe showing a band present in all lanes where 3B chromosome DNA is present.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585268&req=5

pone-0057994-g007: Specific PCR amplification of DNA isolated from flow-sorted FISHIS labeled pasta wheat chromosomes 1A, 6A and 3B.In all Panels: lane M: 50 bp-step ladder; lane WG: whole pasta wheat genomic DNA; lane F-AG: FISHIS labeled flow-sorted whole A-genome chromosomes; lane F-BG: FISHIS labeled flow-sorted whole B-genome chromosomes; lane NF-ALL: whole flow-sorted chromosome complement without FISHIS labeling; lane F-ALL: sorting of the whole FISHIS labeled pasta wheat chromosome complement. Panel a): chromosome 1A amplicons analysis with specific primers from the Xgwm136-1A SSR molecular marker. In lane F-1A, the DNA obtained from 300 FISHIS labeled flow-sorted 1A chromosomes has been PCR amplified showing a band which is only visible where 1A chromosome DNA is present. Panel b): chromosome 6A PCR amplification with primers from the Xgwm169-6A SSR molecular marker. In lane F-6A a specific 6A chromosome band is shown which is absent in all B-genome amplifications. Panel c): chromosome 3B PCR amplification with primers from the Xgwm285-3B SSR molecular marker. In lane F-3B, 300 FISHIS labeled 3B chromosomes have been amplified with a specific probe showing a band present in all lanes where 3B chromosome DNA is present.
Mentions: Single-type FISHIS chromosome and genome fractions along with the total chromosome content of pasta wheat were flow-sorted to evaluate their suitability as a substrate for direct PCR with chromosome-specific primers. We identified and flow-sorted three pasta wheat chromosome fractions which showed very different labeling intensities (Figure 2), that is, 1A, 6A and 3B (Figure S2 and Figure S5). The products from PCR amplifications with primers derived from the chromosome-specific SSR molecular markers Xgwm136, Xgwm169 and Xgwm285 located on chromosomes 1A, 6A and 3B, respectively, are presented and the expected amplicon bands are shown (Figure 7). Wheat A- and B-genome whole-chromosome complements were flow-sorted after FISHIS labeling, proving the effectiveness of the technique in providing a specific PCR probe-related pattern. No sign of contamination or DNA degradation, due to the presence of other chromosome types or to the FISHIS methodology, respectively, was detected (Figure 7).

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