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

Grosso, Valentina; Farina, Anna; Gennaro, Andrea; Giorgi, Debora; Lucretti, Sergio

doi:10.1371/journal.pone.0050151

Cited by 25 publications

(20 citation statements)

References 63 publications

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“…2 e). The chromosomal distribution of (GAA) n repeats in D. breviaristatum chromosome 2V b identified herein is different from the previously known hybridization pattern of D. villosum chromosome 2V [Grosso et al, 2012].…”

Section: Chromosomal Characterizationcontrasting

confidence: 99%

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A Novel Wheat-Dasypyrum breviaristatum Substitution Line with Stripe Rust Resistance

Guang-rong¹,

Zhao²,

Li³

et al. 2014

Cytogenet Genome Res

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The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrumbreviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V^b (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V^b were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V^b of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

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Section: Chromosomal Characterizationcontrasting

confidence: 99%

“…2 ). Meanwhile, the simple sequence repeat (GAA) n can be used as a FISH probe to characterize the individual D. villosum chromosomes [Grosso et al, 2012]. In the present study, we also showed the D. villosum chromosome 2V in the addition line having strong centromeric (GAA) n signals ( fig.…”

Section: Discussionsupporting

confidence: 68%

A Novel Wheat-Dasypyrum breviaristatum Substitution Line with Stripe Rust Resistance

Guang-rong¹,

Zhao²,

Li³

et al. 2014

Cytogenet Genome Res

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“…Flow cytometric data were collected and analyzed using the software package CellQuest Pro v4.01 (BD Bioscience, San Jose, CA). As already described [9], the primary analysis gate was set on a dual parameter dot plot comprising Forward Scatter (FSC) versus FL1H (H = signal height, DAPI fluorescence) to discriminate chromosomes from debris and chromosome aggregates. Sorting windows ( Figure 2 ) were drawn on the fluorescence dot plot of FL1A (A = signal area; DAPI fluorescence) versus FL3H (FITC fluorescence) or FL4H (Cy3 fluorescence).…”

Section: Methodsmentioning

confidence: 99%

“…As of now, methods for chromosome isolation and flow sorting have been reported for 22 plant species including major cereal crops and wild relatives [7], [8], [9]. The separation of a genome into its chromosomal components offers an effective means to generate a full genome sequence of large-genome species, often polyploids such as bread wheat, since it greatly simplifies the process of sequence assembly [8].…”

Section: Introductionmentioning

confidence: 99%

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

et al. 2013

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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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“…The flowcytometric analysis of mitotic chromosomes has been reported in 24 plant species (Doležel et al 2014), including hexaploid and tetraploid wheat and their wild relatives in the genus Aegilops and Dasypyrum (Molnár et al 2011b;Grosso et al 2012). To date, flow cytometric chromosome analysis and sorting has not been reported for the diploid progenitors of bread wheat.…”

Section: Introductionmentioning

confidence: 99%

Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii

et al. 2014

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

Cited by 25 publications

References 63 publications

A Novel Wheat-<b><i>Dasypyrum breviaristatum </i></b>Substitution Line with Stripe Rust Resistance

A Novel Wheat-<b><i>Dasypyrum breviaristatum </i></b>Substitution Line with Stripe Rust Resistance

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii

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