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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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Related in: MedlinePlus

Flow karyotyping and flow sorting of each of the seven D. villosum chromosomes after FISHIS labeling.a) Conventional DNA content-based flow karyotyping resolves only chromosome 6V. b) FISHIS based on the (GAA)7-FITC labeling resolves all seven chromosomes (colored regions) which can be flow-sorted to a purity of 80–95% (purity percentage in Panels). Bar  = 10 µm.
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pone-0057994-g006: Flow karyotyping and flow sorting of each of the seven D. villosum chromosomes after FISHIS labeling.a) Conventional DNA content-based flow karyotyping resolves only chromosome 6V. b) FISHIS based on the (GAA)7-FITC labeling resolves all seven chromosomes (colored regions) which can be flow-sorted to a purity of 80–95% (purity percentage in Panels). Bar  = 10 µm.

Mentions: The next series of experiments involved D. villosum, a wild relative of wheat used in breeding programs as a source of valuable agronomic traits [36]. Characterization of standard chromosome spreads of the D. villosum genome (genome V) using the (GAA)7 probe showed a discrimination of all seven chromosome pairs [9]. For this reason, D. villosum was identified as a suitable candidate for a test of the ultimate resolution power of FISHIS in the flow sorting of chromosomes, namely full discrimination of each chromosome of the genome (Figure 6). The standard mono-parametric flow karyotype of D. villosum comprises four peaks, of which only one is represented by a single chromosome, specifically 6V [9] (Figure 6a). As expected, FISHIS using the (GAA)7-FITC probe generated a dot plot karyotype in which all seven chromosomes could be individually identified and flow-sorted at the levels of purity spanning from 80% to 95% (Figure 6b, colored regions).


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)

Flow karyotyping and flow sorting of each of the seven D. villosum chromosomes after FISHIS labeling.a) Conventional DNA content-based flow karyotyping resolves only chromosome 6V. b) FISHIS based on the (GAA)7-FITC labeling resolves all seven chromosomes (colored regions) which can be flow-sorted to a purity of 80–95% (purity percentage in Panels). Bar  = 10 µm.
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Related In: Results  -  Collection

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

pone-0057994-g006: Flow karyotyping and flow sorting of each of the seven D. villosum chromosomes after FISHIS labeling.a) Conventional DNA content-based flow karyotyping resolves only chromosome 6V. b) FISHIS based on the (GAA)7-FITC labeling resolves all seven chromosomes (colored regions) which can be flow-sorted to a purity of 80–95% (purity percentage in Panels). Bar  = 10 µm.
Mentions: The next series of experiments involved D. villosum, a wild relative of wheat used in breeding programs as a source of valuable agronomic traits [36]. Characterization of standard chromosome spreads of the D. villosum genome (genome V) using the (GAA)7 probe showed a discrimination of all seven chromosome pairs [9]. For this reason, D. villosum was identified as a suitable candidate for a test of the ultimate resolution power of FISHIS in the flow sorting of chromosomes, namely full discrimination of each chromosome of the genome (Figure 6). The standard mono-parametric flow karyotype of D. villosum comprises four peaks, of which only one is represented by a single chromosome, specifically 6V [9] (Figure 6a). As expected, FISHIS using the (GAA)7-FITC probe generated a dot plot karyotype in which all seven chromosomes could be individually identified and flow-sorted at the levels of purity spanning from 80% to 95% (Figure 6b, colored regions).

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