Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of <em>Dasypyrum villosum L</em>. (<em>H. villosa</em>) by a Single DNA Probe

<div><p><em>Dasypyrum villosum</em> (L.) <em>Candargy</em> (sin. <em>Haynaldia villosa</em>) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the <em>Poaceae</em> family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of <em>D. villosum</em> chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of <em>D. villosum</em> chromosomes on slide by fluorescent <em>in situ</em> hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of <em>D. villosum</em> chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.</p> </div

Ideogram of <i>D. villosum</i> chromosomes with the FISH probes labeling patterns.

<p>Ideogram showing the chromosomal distribution of the (GAA)₇ oligonucleotide and of the three repetitive DNA sequences pTa71 (pink bar), pSc119.2 (scarlet bar) and pHv62 (violet bar). GAA bands are shown in black, but the extra yellow band with a star which is detectable only on sorted chromosome 2 V and it is lacking on metaphase spreads. The size of the GAA bands is arbitrarely related to the intensity of the hybridization signals.</p

<i>Dasypyrum villosum</i> FCM karyotyping and chromosome identification after flow sorting.

<p>(a) The flow karyotyping obtained after analysis of DAPI-stained chromosomes show three composite peak (I, II and III) and one peak apart (IV). (b) Sorting regions are drawn around chromosome distribution areas as defined on a fluorescence dot plot of the FL1A (FL1 area, DNA, fluorescence) versus the FSC (chromosome forward scatter). Only three regions, named R1, R2 and R3, were located since the main chromosome distribution did not allowed to focus single chromosomes except a single peak, named region R3. All sorted chromosomes were identify by FISH labelling with (GAA)₇−Cy3 and DAPI staining. In (c-e) images of flow sorted chromosomes from each region are reported. R3 contained a single chromosome (e), identified as 6 V. In R2, an additional GAA band is shown on chromosome 2 V (yellow arrow), which is not present on 2 V metaphase chromosomes and facilitate its discrimination from the 4 V ones in sorted fractions. Scale bar = 10 µm.</p

<i>Dasypyrum villosum</i> metaphases after a “dual blocking steps” cell cycle synchronization procedure.

<p>A high metaphase index (>60%) has been achieved in <i>D. villosum</i> root tips after cell cycle synchronization and metaphase enrichment was induced by incubation in 2 mM hydroxyurea for 18 h, followed by 4 h recovery and a metaphase block after a treatment with 2,5 µM amiprophos-methyl for 2,5 h, followed by overnight incubation in ice water. Feulgen-stained chromosomes are evenly distributed all over the observation field. Scale bar = 100 µm.</p

<i>D. villosum</i> chromosome identification by FISH labelling.

<p>Specific chromosomes and fluorescent bands are indicated by numbers and arrows, respectively. Metaphase chromosomes of: (a) <i>D. villosum</i> Bomarzo accession and (b) <i>D. villosum</i> ‘Greek’ accession, both after ND-FISH (GAA)₇−FITC labeling (<i>green fluorescence</i>) and DAPI staining (<i>blue fluorescence</i>) are shown. All <i>D. villosum</i> Bomarzo chromosomes display a characterizing banding pattern while <i>D. villosum</i> ‘Greek’ accession chromosomes 3 V and 4 V differ in the GAA hybridization pattern, with respect to the Bomarzo accession. (c) Identification of chromosome 1 V in metaphase chromosomes of <i>D. villosum</i> Bomarzo after double target FISH with pTa71-Cy3 (<i>red fluorescence</i>) and (GAA)₇−FITC. (d) Identification of chromosomes 4 V with double target FISH labeling with pSc119.2-Cy3 and (GAA)₇−FITC oligonucleotide to <i>D. villosum</i> Bomarzo metaphase spreads. The pSc119.2 probe hybridized to the 4VS arm only. (e) Metaphase chromosomes of <i>D. villosum</i> Bomarzo after double FISH with pHv62-Cy3 and (GAA)₇−FITC oligonucleotide; chromosome 7 V has been identified by pHv62 labeling. (f) Metaphase chromosomes of <i>T. aestivum</i> CS-6V <i>D. villosum</i> addition line after double FISH labeling of pHv62-Cy3 and (GAA)₇-FITC oligonucleotide. The <i>D. villosum</i> 6 V chromosome added to <i>T. aestivum</i> CS standard complement has been identified by pHv62, a V genome specific probe.</p

FISHIS: Fluorescence <em>In Situ</em> Hybridization in Suspension and Chromosome Flow Sorting Made Easy

<div><p>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 <i>In situ</i> 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 (<i>T. durum</i> L.) and bread (<i>T. aestivum</i> L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, <i>Dasypyrum villosum</i> (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.</p> </div

(GAA)₇-FITC labeling and FISHIS-based flow-sorting of bread wheat chromosomes (<i>T. aestivum</i> cv Chinese Spring double ditelosomic line CSdDt5A).

<p>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.</p

Flow karyotyping and flow sorting of each of the seven <i>D. villosum</i> chromosomes after FISHIS labeling.

<p>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.</p

High purity flow sorting of (AG)₁₂-Cy3 labeled 5B and 3B chromosomes from bread wheat line CSdDt5A.

<p>Two sorting regions can be drawn on a DAPI-DNA fluorescence versus a (AG)₁₂-Cy3 FISHIS fluorescence dot plot, which enclose chromosomes 5B and 3B at a purity of 91 and 99%, respectively. Chromosome arms 5AS and 5AL are also bordered to show the high selectivity for chromosome labeling of the probe (AG)₁₂, which do not alter the discrimination of the other remaining chromosomes and chromosome arms, as shown for (GAA)₇ labeling (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057994#pone-0057994-g004" target="_blank"><b>Figure 4</b></a>). Bar  = 10 µm.</p

FISHIS of pasta wheat cv Creso chromosome suspensions.

<p>a) Chromosome suspensions hybridized with (GAA)₇-FITC; b) flow-sorted chromosomes 3B, 4B and 5B following hybridization with (AG)₁₂-Cy3; c) (GAA)₇-FITC and (AAT)₇-Cy3 dual labelled chromosomes and nuclei; d) pasta wheat chromosomes and a nucleus after (GAA)₇-FITC and (AAC)₅-Cy3 dual labeling. Bar  = 10 µm.</p