Flow sorting and molecular cytogenetic identification of individual chromosomes of Dasypyrum villosum L. (H. villosa) by a single DNA probe

Abstract

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum 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.

Figure 1. D. villosum chromosome identification by FISH labelling.

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

Figure 2. Ideogram of D. villosum chromosomes with the FISH probes labeling patterns.

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.

Figure 3. Dasypyrum villosum metaphases after a “dual blocking steps” cell cycle synchronization procedure.

A high metaphase index (>60%) has been achieved in D. villosum 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.

Figure 4. Dasypyrum villosum FCM karyotyping and chromosome identification after flow sorting.

(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.