Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

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 pre pared by mechanical homogenization of synchronized root tips after mild fix ation with formaldehyde. Histograms of relative fluorescence intensity (flo w karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat now karyotyp e was determined for the first time. Only chromosome 3B could be discrimina ted on flow karyotypes of wheat lines with standard karyotype. Remaining ch romosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic e valuation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7 BL could be sorted in two wheat cultivars at similar purity, indicating a p otential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fraction s and suitability of flow-sorted chromosomes for physical mapping and for c onstruction of small-insert DNA libraries. Sorted chromosomes were also fou nd suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-inser t chromosome-specific DNA libraries in wheat. The availability of such libr aries would greatly simplify the analysis of the complex wheat genome.