The large bread wheat genome (1C ∼ 17 Gbp) contains a preponderance of repetitive DNA and the species is polyploid. These characteristics together serve to hamper the molecular analysis of the wheat genome. Its complexity can, however, be reduced by using flow cytometry to isolate individual chromosomes, and these can be exploited to construct chromosome-specific BAC libraries. Such libraries simplify the task of physical map construction, positional cloning and the targeted development of genetic markers. Rapid improvements in the efficiency and cost of DNA sequencing provide an opportunity to contemplate sequencing the wheat genome by preparing sequence-ready physical maps for each chromosome or chromosome arm in turn. The quality of the chromosome-specific libraries depends on their chromosome coverage and the mean insert size. First-generation libraries suffered from a relatively low mean insert size, but improvements to the protocol have generated a second wave of libraries with a significantly increased mean insert size and better chromosome coverage. Each chromosome (arm)-specific library is composed of a manageable number of clones, and so represents a practical tool in the area of wheat genomics.

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.

Although some species of the tribe Triticeae possess small genomes, the genomes of barley (~5000 Mb/1C), rye (~8000 Mb/1C), durum wheat (~13,000 Mb/1C) and bread wheat (~17,000 Mb/1C) are complex, consisting mainly of various classes of repetitive DNA sequences.In addition, the recent evolution of wheat involved two episodes of polyploidization giving rise to progenitors of allotetraploid durum wheat and allohexaploid bread wheat.These features hamper physical mapping and gene cloning.Purification of individual chromosomes by flow cytometry can simplify these tasks by providing small and defined genome fractions.Unfortunately, only one chromosome can be discriminated and sorted in each of the four species due to small differences in DNA content.We have demonstrated that this problem can be overcome by means of cytogenetic stocks from which particular chromosomes and chromosome arms can be discriminated and sorted using flow cytometry.The use of telosomic lines of bread wheat facilitates sorting 40 out of 42 chromosome arms.The remaining two arms, 3BL and 5BL, can be purified as isochromosomes.In durum wheat, the use of telosomic lines facilitates sorting any of the 28 chromosome arms.Flow cytometric fractionation of the genomes of barley and rye relies on the capacity of polyploid wheat to maintain chromosomes and chromosome arms of other genomes of Triticeae as addition lines.Flow cytometric analysis of wheat-barley telosome addition lines revealed that they may be used to sort any of the fourteen barley chromosome arms.A similar approach can be used to fractionate the genome of rye.We have found that rye chromosomes 2R-7R, which cannot be sorted from a standard karyotype, could be discriminated and sorted from individual wheat-rye addition lines.To conclude, we have developed a universal flow-based platform that is suitable for dissecting the genomes of wheat, barley and rye.Because the DNA of sorted chromosomes is intact, they are suitable for a range of applications including construction of subgenomic DNA libraries and molecular cytogenetic mapping.