FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.
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 discre...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3585268?pdf=render |
| _version_ | 1811333952265256960 |
|---|---|
| author | Debora Giorgi Anna Farina Valentina Grosso Andrea Gennaro Carla Ceoloni Sergio Lucretti |
| author_facet | Debora Giorgi Anna Farina Valentina Grosso Andrea Gennaro Carla Ceoloni Sergio Lucretti |
| author_sort | Debora Giorgi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T17:00:30Z |
| format | Article |
| id | doaj.art-83d68edf684148d8b193f70b3022fb92 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T17:00:30Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-83d68edf684148d8b193f70b3022fb922022-12-22T02:38:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5799410.1371/journal.pone.0057994FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.Debora GiorgiAnna FarinaValentina GrossoAndrea GennaroCarla CeoloniSergio LucrettiThe 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.http://europepmc.org/articles/PMC3585268?pdf=render |
| spellingShingle | Debora Giorgi Anna Farina Valentina Grosso Andrea Gennaro Carla Ceoloni Sergio Lucretti FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS ONE |
| title | FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. |
| title_full | FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. |
| title_fullStr | FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. |
| title_full_unstemmed | FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. |
| title_short | FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. |
| title_sort | fishis fluorescence in situ hybridization in suspension and chromosome flow sorting made easy |
| url | http://europepmc.org/articles/PMC3585268?pdf=render |
| work_keys_str_mv | AT deboragiorgi fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy AT annafarina fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy AT valentinagrosso fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy AT andreagennaro fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy AT carlaceoloni fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy AT sergiolucretti fishisfluorescenceinsituhybridizationinsuspensionandchromosomeflowsortingmadeeasy |