Limits...
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.

Show MeSH

Related in: MedlinePlus

(GAA)7-FITC labeling and FISHIS-based flow-sorting of bread wheat chromosomes (T. aestivum cv Chinese Spring double ditelosomic line CSdDt5A).a) FISHIS allows the discrimination between homeologous genomes A and D (within BOX1) and B-genome; b) the increasing of the instrument sensibility towards lower fluorescence signals permits an easy flow-sorting of chromosome 4A (colored region); c) by doubling the signal amplification, all the D-genome chromosomes and the chromosome 1A can be confined into specific sorting regions (color-marked areas). Chromosomes 2A, 6A, and chromosome arms 5AS and 5AL were sortable to a high level of purity (purity percentage in Panels). Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585268&req=5

pone-0057994-g004: (GAA)7-FITC labeling and FISHIS-based flow-sorting of bread wheat chromosomes (T. aestivum cv Chinese Spring double ditelosomic line CSdDt5A).a) FISHIS allows the discrimination between homeologous genomes A and D (within BOX1) and B-genome; b) the increasing of the instrument sensibility towards lower fluorescence signals permits an easy flow-sorting of chromosome 4A (colored region); c) by doubling the signal amplification, all the D-genome chromosomes and the chromosome 1A can be confined into specific sorting regions (color-marked areas). Chromosomes 2A, 6A, and chromosome arms 5AS and 5AL were sortable to a high level of purity (purity percentage in Panels). Bar  = 10 µm.

Mentions: In bread wheat, the standard mono-parametric flow karyotype comprehends four main peaks, but only one of these contains a single chromosome, namely 3B [10]. With FISHIS, for cv Provinciale or for line CSdDt5A (a CS cytogenetic stock previously used for the isolation and sequencing of chromosome 5A arms [35]), it was possible to discriminate between all three homeologous genomes (Figure 4a), and inside each genome region, specific individual chromosomes were characterized and located. Figure 4 illustrates a detailed analysis of FISHIS in CSdDt5A, and how, by optimizing the flow cytometer set up, it becomes possible to identify specific components of the wheat genome (Figure 4a). As previously seen for pasta wheat, the B-genome of bread wheat can be easily recognized within the upper region of the dot plot. After optimization, it was possible to sort chromosome 4A to a purity of above 93% (Figure 4b, region in color). Further magnification of the relevant region in the dot plot demonstrates discrimination of all A- and D-genome chromosomes, with the exception of 1A (Figure 4c). Chromosomes 2A and 6A were subsequently sorted to high purity levels (>92% and >91%, respectively; Figure 4c). It should be noted that a further advantage of FISHIS is that chromosome purity assessment, conventionally done by an overnight FISH analysis using the sorted fraction [14], is made in real time, since chromosomes are labeled already, which means that the sorting parameters can be immediately optimized. This new feature could facilitate the standard chromosome approach, expediting whole chromosomes and single arms spotting (see telocentrics 5AS and 5AL: Figure 4c).


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 bread wheat chromosomes (T. aestivum cv Chinese Spring double ditelosomic line CSdDt5A).a) FISHIS allows the discrimination between homeologous genomes A and D (within BOX1) and B-genome; b) the increasing of the instrument sensibility towards lower fluorescence signals permits an easy flow-sorting of chromosome 4A (colored region); c) by doubling the signal amplification, all the D-genome chromosomes and the chromosome 1A can be confined into specific sorting regions (color-marked areas). Chromosomes 2A, 6A, and chromosome arms 5AS and 5AL were sortable to a high level of purity (purity percentage in Panels). Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057994-g004: (GAA)7-FITC labeling and FISHIS-based flow-sorting of bread wheat chromosomes (T. aestivum cv Chinese Spring double ditelosomic line CSdDt5A).a) FISHIS allows the discrimination between homeologous genomes A and D (within BOX1) and B-genome; b) the increasing of the instrument sensibility towards lower fluorescence signals permits an easy flow-sorting of chromosome 4A (colored region); c) by doubling the signal amplification, all the D-genome chromosomes and the chromosome 1A can be confined into specific sorting regions (color-marked areas). Chromosomes 2A, 6A, and chromosome arms 5AS and 5AL were sortable to a high level of purity (purity percentage in Panels). Bar  = 10 µm.
Mentions: In bread wheat, the standard mono-parametric flow karyotype comprehends four main peaks, but only one of these contains a single chromosome, namely 3B [10]. With FISHIS, for cv Provinciale or for line CSdDt5A (a CS cytogenetic stock previously used for the isolation and sequencing of chromosome 5A arms [35]), it was possible to discriminate between all three homeologous genomes (Figure 4a), and inside each genome region, specific individual chromosomes were characterized and located. Figure 4 illustrates a detailed analysis of FISHIS in CSdDt5A, and how, by optimizing the flow cytometer set up, it becomes possible to identify specific components of the wheat genome (Figure 4a). As previously seen for pasta wheat, the B-genome of bread wheat can be easily recognized within the upper region of the dot plot. After optimization, it was possible to sort chromosome 4A to a purity of above 93% (Figure 4b, region in color). Further magnification of the relevant region in the dot plot demonstrates discrimination of all A- and D-genome chromosomes, with the exception of 1A (Figure 4c). Chromosomes 2A and 6A were subsequently sorted to high purity levels (>92% and >91%, respectively; Figure 4c). It should be noted that a further advantage of FISHIS is that chromosome purity assessment, conventionally done by an overnight FISH analysis using the sorted fraction [14], is made in real time, since chromosomes are labeled already, which means that the sorting parameters can be immediately optimized. This new feature could facilitate the standard chromosome approach, expediting whole chromosomes and single arms spotting (see telocentrics 5AS and 5AL: Figure 4c).

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