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.

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

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Bedbrook, J. R., J. Jones, M. O'Dell, R. D. Thompson and R. B. Flavell, 1980. A molecular description of telomeric heterochromatin in Secale species. Cell 19: 545–560. PubMed

Bennett, M. D., and I. J. Leitch, 1995. Nuclear-DNA amounts in angiosperms. Ann. Bot. 76: 113–176.

Cenci, A., N. Chantret, X. Kong, Y. Gu, O. D. Anderson et al., 2003. Construction and characterization of a half million clone BAC library of durum wheat (Triticum turgidum ssp. durum). Theor. Appl. Genet. 107: 931–939. PubMed

Doležel, J., P. Binarová and S. Lucretti, 1989. Analysis of nuclear DNA content in plant cells by flow cytometry. Biol. Plant. 31: 113–120.

Doležel, J., S. Lucretti and I. Schubert, 1994. Plant chromosome analysis and sorting by flow cytometry. Crit. Rev. Plant Sci. 13: 275–309.

Doležel, J., M. Kubaláková, J. Bartoš and J. Macas, 2004. Flow cytogenetics and plant genome mapping. Chromosome Res. 12: 77–91. PubMed

Erayman, M., D. Sandhu, D. Sidhu, M. Dilbirligi, P. S. Baenziger et al., 2004. Demarcating the gene-rich regions of wheat genome. Nucleic Acids Res. 32: 3546–3565. PubMed PMC

Faris, J. D., J. P. Fellers, S. A. Brooks and B. S. Gill, 2003. A bacterial artificial chromosome contig spanning the major domestication locus Q in wheat and identification of a candidate gene. Genetics 164: 311–321. PubMed PMC

Friebe, B., and B. S. Gill, 1994. C-band polymorphism and structural rearrangements detected in common wheat (Triticum aestivum). Euphytica 78: 1–5.

Fukui, K., N. Ohmido and G. S. Khush, 1994. Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization. Theor. Appl. Genet. 87: 893–899. PubMed

Gamborg, O. L., and L. R. Wetter, 1975 Plant Tissue Culture Methods. National Research Council of Canada, Saskatoon, SK, Canada.

Gill, B. S., R. Appels, A.-M. Botha-Oberholster, C. R. Buell, J. L. Bennetzen et al., 2004. A workshop report on wheat genome sequencing: international genome research on wheat consortium. Genetics 168: 1087–1096. PubMed PMC

Gribble, S. M., H. Fiegler, D. C. Burford, E. Prigmore, F. Yang et al., 2004. Applications of combined DNA microarray and chromosome sorting technologies. Chromosome Res. 12: 35–43. PubMed

Gu, Y. Q., D. Coleman-Derr, X. Y. Kong and O. D. Anderson, 2004. Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes. Plant Physiol. 135: 459–470. PubMed PMC

Huang, L., S. A. Brooks, W. Li, J. P. Fellers, H. N. Trick et al., 2003. Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploidy genome of bread wheat. Genetics 164: 655–664. PubMed PMC

Huang, S., A. Sirikhachornkit, X. J. Su, J. Faris, B. Gill et al., 2002. Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat. Proc. Natl. Acad. Sci. USA 99: 8133–8138. PubMed PMC

Janda, J., J. Bartoš, J. Šafář, M. Kubaláková, M. Valárik et al., 2004. Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat. Theor. Appl. Genet. 109: 1337–1345. PubMed

Joppa, L. R., 1993. Chromosome engineering in tetraploid wheat. Crop Sci. 33: 908–913.

Joppa, L. R., and N. D. Williams, 1988. Langdon durum disomic substitution lines and aneuploid analysis in tetraploid wheat. Genome 30: 222–228.

Kejnovský, E., J. Vrána, S. Matsunaga, P. Souček, J. Široký et al., 2001. Localization of male-specifically expressed MROS genes of Silene latifolia by PCR on flow-sorted sex chromosomes and autosomes. Genetics 158: 1269–1277. PubMed PMC

Kihara, H., 1944. Discovery of the DD-analyzer, one of the ancestors of vulgare wheats. Agric. Hortic. 19: 889–890.

Kubaláková, M., J. Macas and J. Doležel, 1997. Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS. Theor. Appl. Genet. 94: 758–763.

Kubaláková, M., J. Vrána, J. Číhalíková, H. Šimková and J. Doležel, 2002. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 104: 1362–1372. PubMed

Kubaláková, M., M. Valárik, J. Bartoš, J. Vrána, J. Číhalíková et al., 2003. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46: 893–905. PubMed

Lander, E. S., L. M. Linton, B. Birren, C. Nusbaum, M. C. Zody et al., 2001. Initial sequencing and analysis of the human genome. Nature 409: 860–921. PubMed

Luo, M. C., C. Thomas, F. M. You, J. Hsiao, O. Y. Shu et al., 2003. High-throughput fingerprinting of bacterial artificial chromosomes using the SNaPshot labeling kit and sizing of restriction fragments by capillary electrophoresis. Genomics 82: 378–389. PubMed

Lysák, M. A., J. Číhalíková, M. Kubaláková, H. Šimková, G. Künzel et al., 1999. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Res. 7: 431–444. PubMed

McFadden, E. S., and E. R. Sears, 1946. The origin of Triticum spelta and its free-threshing hexaploid relatives. J. Hered. 37: 81–89. PubMed

Mukai, Y., Y. Nakahara and M. Yamamoto, 1993. Simultaneous discrimination of the 3 genomes in hexaploid wheat by multicolour fluorescence in situ hybridisation using total genomic and highly repeated DNA probes. Genome 36: 489–494. PubMed

Nagaki, K., H. Tsujimoto, K. Isono and T. Sasakuma, 1995. Molecular characterization of a tandem repeat, Afa family, and its distribution among Triticeae. Genome 38: 479–486. PubMed

Neumann, P., D. Požárková, J. Vrána, J. Doležel and J. Macas, 2002. Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.). Chromosome Res. 10: 63–71. PubMed

Nishikawa, K., A. Takagi, T. Ban, H. Ohtsuka and Y. Furuta, 1986. Spontaneous reciprocal translocation in cultivated form of emmer wheat. Jpn. J. Genet. 61: 361–370.

Pedersen, C., and P. Langridge, 1997. Identification of the entire chromosome complement of bread wheat by two-colour FISH. Genome 40: 589–593. PubMed

Peterson, D. G., S. R. Schulze, E. B. Sciara, S. A. Lee, J. E. Bowers et al., 2002. Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. Genome Res. 12: 795–807. PubMed PMC

Požárková, D., A. Koblížková, B. Román, A. M. Torres, S. Lucretti et al., 2002. Development and characterization of microsatellite markers from chromosome 1-specific DNA libraries of Vicia faba. Biol. Plant. 45: 337–345.

Román, B., Z. Satovic, D. Požárková, J. Macas, J. Doležel et al., 2004. Development of a composite map in Vicia faba, breeding applications and future prospects. Theor. Appl. Genet. 108: 1079–1088. PubMed

Šafář, J., J. Bartoš, J. Janda, A. Bellec, M. Kubaláková et al., 2004. Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat. Plant J. 39: 960–968. PubMed

Sears, E. R., 1966 Nullisomic-tetrasomic combination in hexaploid wheat, pp. 29–45 in Chromosome Manipulation and Plant Genetics, edited by R. Riley and K. R. Lewis. Oliver & Boyd, Edinburgh.

Sears, E. R., 1969. Wheat cytogenetics. Annu. Rev. Genet. 3: 451–468.

Thangavelu, M., A. B. James, A. Bankier, G. J. Bryan, P. H. Dear et al., 2003. HAPPY mapping in a plant genome: reconstruction and analysis of a high-resolution physical map of a 1.9 Mbp region of Arabidopsis thaliana chromosome 4. Plant Biotech. J. 1: 23–31. PubMed

Tsunewaki, K., 1963. An emmer wheat with 15 chromosome pairs. Can. J. Genet. Cytol. 5: 462–466.

Valárik, M., H. Šimková, E. Hřibová, J. Šafář, M. Doleželová et al., 2002. Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.). Chromosome Res. 10: 89–100. PubMed

Valárik, M., J. Bartoš, P. Kovářová, M. Kubaláková, J. H. de Jong et al., 2004. High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant J. 37: 940–950. PubMed

Venora, G., S. Blangiforti, M. R. Castiglione, D. Pignone, F. Losavio et al., 2002. Chromatin organisation and computer aided karyotyping of Triticum durum Desf. cv. Timilia. Caryologia 55: 91–98.

Vláčilová, K., D. Ohri, J. Vrána, J. Číhalíková, M. Kubaláková et al., 2002. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Res. 10: 695–706. PubMed

Vrána, J., M. Kubaláková, H. Šimková, J. Číhalíková, M. A. Lysák et al., 2000. Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033–2041. PubMed PMC

Wicker, T., N. Yahiaoui, R. Guyot, E. Schlagenhauf, Z. D. Liu et al., 2003. Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and A(m) genomes of wheat. Plant Cell 15: 1186–1197. PubMed PMC

Yan, L., A. Loukoianov, G. Tranquilli, M. Helguera, T. Fahima et al., 2003. Positional cloning of the wheat vernalization gene Vrn1. Proc. Natl. Acad. Sci. USA 100: 6263–6268. PubMed PMC

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