The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.

Laboratory of Molecular Cytogenetics and Cytometry Institute of Experimental Botany CZ 77200 Olomouc Czech Republic

Zobrazit více v PubMed

Cytometry. 1992;13(5):469-77 PubMed

Mamm Genome. 1999 Jul;10(7):687-91 PubMed

Genomics. 1992 Jul;13(3):718-25 PubMed

Mol Gen Genet. 1994 Mar;242(5):551-8 PubMed

Chromosome Res. 1999;7(6):431-44 PubMed

J Mol Evol. 1998 Aug;47(2):183-9 PubMed

Chromosome Res. 1993 Jul;1(2):107-15 PubMed

Eur J Hum Genet. 1997 Sep-Oct;5(5):253-65 PubMed

Genetics. 1998 Aug;149(4):2007-23 PubMed

Chromosome Res. 1996 Nov;4(7):531-9 PubMed

Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1063-7 PubMed

Proc Natl Acad Sci U S A. 1979 Mar;76(3):1382-4 PubMed

Genetics. 1996 Jun;143(2):1001-12 PubMed

Cytometry. 1990;11(1):208-18 PubMed

Genome. 1997 Oct;40(5):589-93 PubMed

Cytometry. 1982 Nov;3(3):145-54 PubMed

Genome. 1997 Oct;40(5):633-8 PubMed

Chromosome Res. 1997 Apr;5(2):102-8 PubMed

Philos Trans R Soc Lond B Biol Sci. 1976 May 27;274(933):227-74 PubMed

Genome. 1993 Apr;36(2):387-90 PubMed

Genome. 1996 Aug;39(4):697-703 PubMed

Anim Genet. 1992;23(3):203-10 PubMed

Nucleic Acids Res. 1992 Apr 25;20(8):1897-901 PubMed

Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69

Flow Cytometric Analysis and Sorting of Plant Chromosomes

The wheat stem rust resistance gene Sr43 encodes an unusual protein kinase

Flow karyotyping of wheat-Aegilops additions facilitate dissecting the genomes of Ae. biuncialis and Ae. geniculata into individual chromosomes

Isolation and Sequencing of Chromosome Arm 7RS of Rye, Secale cereale

Identification, High-Density Mapping, and Characterization of New Major Powdery Mildew Resistance Loci From the Emmer Wheat Landrace GZ1

Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62

In-Depth Sequence Analysis of Bread Wheat VRN1 Genes

Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences

Chromosome analysis and sorting

Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins

A membrane-bound ankyrin repeat protein confers race-specific leaf rust disease resistance in wheat

Multiple origins of Indian dwarf wheat by mutations targeting the TREE domain of a GSK3-like kinase for drought tolerance, phosphate uptake, and grain quality

Structural Variations Affecting Genes and Transposable Elements of Chromosome 3B in Wheats

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp

Aegilops umbellulata introgression carrying leaf rust and stripe rust resistance genes Lr76 and Yr70 located to 9.47-Mb region on 5DS telomeric end through a combination of chromosome sorting and sequencing

Stem rust resistance in wheat is suppressed by a subunit of the mediator complex

Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars

Identification of a Dominant Chlorosis Phenotype Through a Forward Screen of the Triticum turgidum cv. Kronos TILLING Population

Fine Mapping of Lr49 Using 90K SNP Chip Array and Flow-Sorted Chromosome Sequencing in Wheat