The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany 78371 Olomouc Czech Republic

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Alm V, Fang C, Busso CS, Devos KM, Vollan K, Grieg Z, Rognli OA. (2003) A linkage map of meadow fescue (Festuca pratensis Huds.) and comparative mapping with other Poaceae species. Theor Appl Genet 108: 25–40 PubMed

Baird JH, Kopecký D, Lukaszewski AJ, Green RL, Bartoš J, Doležel J. (2012) Genetic diversity of turf-type tall fescue using diversity arrays technology. Crop Sci 52: 408–412

Bartoš J, Sandve SR, Kölliker R, Kopecký D, Christelová P, Stočes S, Østrem L, Larsen A, Kilian A, Rognli OA, et al. (2011) Genetic mapping of DArT markers in the Festuca-Lolium complex and their use in freezing tolerance association analysis. Theor Appl Genet 122: 1133–1147 PubMed

Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E. (2008) Fast unfolding of communities in large networks. J Stat Mech 2008: P10008

Brandes A, Röder MS, Ganal MW. (1995) Barley telomeres are associated with two different types of satellite DNA sequences. Chromosome Res 3: 315–320 PubMed

Byrne S, Panitz F, Hedegaard J, Bendixen C, Studer B, Farrell JD, Swain SC, Armstead I, Caccamo M, Asp T (2011) De novo genome sequencing of perennial ryegrass (Lolium perenne). In S Barth, D Milbourne, eds, Abstracts of the EUCARPIA 29th Fodder Crops and Amenity Grasses Section Meeting. Teagasc, Dublin, p 23

Chalupska D, Lee HY, Faris JD, Evrard A, Chalhoub B, Haselkorn R, Gornicki P. (2008) Acc homoeoloci and the evolution of wheat genomes. Proc Natl Acad Sci USA 105: 9691–9696 PubMed PMC

Choulet F, Wicker T, Rustenholz C, Paux E, Salse J, Leroy P, Schlub S, Le Paslier M-C, Magdelenat G, Gonthier C, et al. (2010) Megabase level sequencing reveals contrasted organization and evolution patterns of the wheat gene and transposable element spaces. Plant Cell 22: 1686–1701 PubMed PMC

Doležel J, Bartoš J, Voglmayr H, Greilhuber J. (2003) Nuclear DNA content and genome size of trout and human. Cytometry A 51: 127–128, author reply 129 PubMed

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

Doležel J, Číhalíková J, Lucretti S. (1992) A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta 188: 93–98 PubMed

Doležel J, Šimková H, Kubaláková M, Šafář J, Suchánková P, Číhalíková J, Bartoš J, Valárik M (2009) Chromosome genomics in the Triticeae. In C Feuillet, GJ Muehlbauer, eds, Genetics and Genomics of the Triticeae. Plant Genetics and Genomics: Crops and Models 7. Springer Science+Business Media, New York, pp 285–316

Donnison IS, O’Sullivan DM, Thomas A, Canter P, Moore B, Armstead I, Thomas H, Edwards KJ, King IP. (2005) Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines. Theor Appl Genet 110: 846–851 PubMed

Egan AN, Schlueter J, Spooner DM. (2012) Applications of next-generation sequencing in plant biology. Am J Bot 99: 175–185 PubMed

Fukui K, Kamisugi Y, Sakai F. (1994) Physical mapping of 5S rDNA loci by direct-cloned biotinylated probes in barley chromosomes. Genome 37: 105–111 PubMed

Gale MD, Devos KM. (1998) Plant comparative genetics after 10 years. Science 282: 656–659 PubMed

Gamborg OL, Wetter LR (1975) Plant Tissue Culture Methods. National Research Council of Canada, Saskatoon, Canada

Gerlach WL, Bedbrook JR. (1979) Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7: 1869–1885 PubMed PMC

Hernandez P, Martis M, Dorado G, Pfeifer M, Gálvez S, Schaaf S, Jouve N, Šimková H, Valárik M, Doležel J, et al. (2012) Next-generation sequencing and syntenic integration of flow-sorted arms of wheat chromosome 4A exposes the chromosome structure and gene content. Plant J 69: 377–386 PubMed

Hřibová E, Dolezelová M, Town CD, Macas J, Doležel J. (2007) Isolation and characterization of the highly repeated fraction of the banana genome. Cytogenet Genome Res 119: 268–274 PubMed

Jaccoud D, Peng K, Feinstein D, Kilian A. (2001) Diversity arrays: a solid state technology for sequence information independent genotyping. Nucleic Acids Res 29: E25. PubMed PMC

Katsiotis A, Hagidimitriou M, Heslop-Harrison JS. (1997) The close relationship between the A and B genomes in Avena L. (Poaceae) determined by molecular cytogenetic analysis of total genomic, tandemly and dispersed repetitive DNA sequences. Ann Bot (Lond) 79: 103–109

Kopecký D, Bartoš J, Christelová P, Černoch V, Kilian A, Doležel J. (2011) Genomic constitution of Festuca × Lolium hybrids revealed by the DArTFest array. Theor Appl Genet 122: 355–363 PubMed

Kopecký D, Bartoš J, Lukaszewski AJ, Baird JH, Černoch V, Kölliker R, Rognli OA, Blois H, Caig V, Lübberstedt T, et al. (2009) Development and mapping of DArT markers within the Festuca-Lolium complex. BMC Genomics 10: 473. PubMed PMC

Kopecký D, Havránková M, Loureiro J, Castro S, Lukaszewski AJ, Bartoš J, Kopecká J, Doležel J. (2010) Physical distribution of homoeologous recombination in individual chromosomes of Festuca pratensis in Lolium multiflorum. Cytogenet Genome Res 129: 162–172 PubMed

Kopecký D, Lukaszewski AJ, Doležel J. (2008a) Cytogenetics of Festulolium (Festuca × Lolium hybrids). Cytogenet Genome Res 120: 370–383 PubMed

Kopecký D, Lukaszewski AJ, Doležel J. (2008b) Meiotic behaviour of individual chromosomes of Festuca pratensis in tetraploid Lolium multiflorum. Chromosome Res 16: 987–998 PubMed

Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19: 1639–1645 PubMed PMC

Kubaláková M, Valárik M, Barto J, Vrána J, Číhalíková J, Molnár-Láng M, Doležel J. (2003) Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46: 893–905 PubMed

Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, et al. (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1: 18. PubMed PMC

Mandáková T, Lysák MA. (2008) Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae). Plant Cell 20: 2559–2570 PubMed PMC

Martienssen RA, Rabinowicz PD, O’Shaughnessy A, McCombie WR. (2004) Sequencing the maize genome. Curr Opin Plant Biol 7: 102–107 PubMed

Masoudi-Nejad A, Nasuda S, McIntosh RA, Endo TR. (2002) Transfer of rye chromosome segments to wheat by a gametocidal system. Chromosome Res 10: 349–357 PubMed

Mayer KFX, Martis M, Hedley PE, Simková H, Liu H, Morris JA, Steuernagel B, Taudien S, Roessner S, Gundlach H, et al. (2011) Unlocking the barley genome by chromosomal and comparative genomics. Plant Cell 23: 1249–1263 PubMed PMC

Mayer KF, Taudien S, Martis M, Simkova H, Suchankova P, Gundlach H, Wicker T, Petzold A, Felder M, Steuernagel B, et al. (2009) Gene content and virtual gene order of barley chromosome 1H. Plant Physiol 151: 496–505 PubMed PMC

Mayer KF, Waugh R, Brown JW, Schulman A, Langridge P, Platzer M, Fincher GB, Muehlbauer GJ, Sato K, Close TJ, et al. (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491: 711–716 PubMed

Naranjo T, Roca A, Goicoecha PG, Giraldz R. (1987) Arm homoeology of wheat and rye chromosomes. Genome 29: 873–882

Novák P, Neumann P, Macas J. (2010) Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data. BMC Bioinformatics 11: 378. PubMed PMC

Paux E, Sourdille P, Salse J, Saintenac C, Choulet F, Leroy P, Korol A, Michalak M, Kianian S, Spielmeyer W, et al. (2008) A physical map of the 1-gigabase bread wheat chromosome 3B. Science 322: 101–104 PubMed

Pedrosa-Harand A, Kami J, Gepts P, Geffroy V, Schweizer D. (2009) Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species. Chromosome Res 17: 405–417 PubMed

Peterson DG, Wessler SR, Paterson AH. (2002) Efficient capture of unique sequences from eukaryotic genomes. Trends Genet 18: 547–550 PubMed

Pfeifer M, Martis M, Asp T, Mayer KFX, Lübberstedt T, Byrne S, Frei U, Studer B. (2013) The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics. Plant Physiol 161: 571–582 PubMed PMC

Polok K (2007) Molecular Evolution of the Genus Lolium L. Studio Poligrafii Komputerowej, Olsztyn, Poland

Rabinowicz PD, Schutz K, Dedhia N, Yordan C, Parnell LD, Stein L, McCombie WR, Martienssen RA. (1999) Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat Genet 23: 305–308 PubMed

Rayburn AL, Gill BS. (1986) Molecular identification of the D-genome chromosomes of beat. J Hered 77: 253–255

Šafář J, Šimková H, Kubaláková M, Číhalíková J, Suchánková P, Bartoš J, Doležel J. (2010) Development of chromosome-specific BAC resources for genomics of bread wheat. Cytogenet Genome Res 129: 211–223 PubMed

Schwarzacher T, Heslop-Harrison P (2000) Practical in Situ Hybridization. BIOS Scientific Publishers, Oxford

Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS. (1989) In situ localization of parental genomes in a wide hybrid. Ann Bot (Lond) 64: 315–324

Sharma S, Raina SN. (2005) Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes. Cytogenet Genome Res 109: 15–26 PubMed

Šimková H, Svensson JT, Condamine P, Hřibová E, Suchánková P, Bhat PR, Bartoš J, Šafář J, Close TJ, Doležel J. (2008) Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genomics 9: 294. PubMed PMC

Sonnhammer ELL, Durbin R. (1995) A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167: GC1–GC10 PubMed

Studer B, Byrne S, Nielsen RO, Panitz F, Bendixen C, Islam MS, Pfeifer M, Lübberstedt T, Asp T. (2012) A transcriptome map of perennial ryegrass (Lolium perenne L.). BMC Genomics 13: 140. PubMed PMC

Suchánková P, Kubaláková M, Kovárová P, Bartoš J, Číhalíková J, Molnár-Láng M, Endo TR, Doležel J. (2006) Dissection of the nuclear genome of barley by chromosome flow sorting. Theor Appl Genet 113: 651–659 PubMed

Thiel T, Graner A, Waugh R, Grosse I, Close TJ, Stein N. (2009) Evidence and evolutionary analysis of ancient whole-genome duplication in barley predating the divergence from rice. BMC Evol Biol 9: 209. PubMed PMC

Thomas HM, Morgan WG, Meredith MR, Humphreys MW, Thomas H, Leggett JM. (1994) Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization. Theor Appl Genet 88: 909–913 PubMed

Tomaszewski C, Byrne S, Foito A, Kildea S, Kopecký D, Doležel J, Heslop-Harrison JS, Stewart D, Barth S. (2012) A genetic linkage map of an F2 perennial ryegrass population highly enriched with DArT markers suitable for mapping of crown rust resistence QTL. Plant Breed 131: 345–349

Tsujimoto H, Mukai Y, Akagawa K, Nagaki K, Fujigaki J, Yamamoto M, Sasakuma T. (1997) Identification of individual barley chromosomes based on repetitive sequences: conservative distribution of Afa-family repetitive sequences on the chromosomes of barley and wheat. Genes Genet Syst 72: 303–309 PubMed

Vigeland MD, Spannagl M, Asp T, Paina C, Rudi H, Rognli OA, Fjellheim S, Sandve SR. (2013) Evidence for adaptive evolution of low-temperature stress response genes in a Pooideae grass ancestor. New Phytol 199: 1060–1068 PubMed PMC

Vitulo N, Albiero A, Forcato C, Campagna D, Dal Pero F, Bagnaresi P, Colaiacovo M, Faccioli P, Lamontanara A, Šimková H, et al. (2011) First survey of the wheat chromosome 5A composition through a next generation sequencing approach. PLoS ONE 6: e26421. PubMed PMC

Vrána J, Kubaláková M, Simková H, Cíhalíková J, Lysák MA, Doležel J. (2000) Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033–2041 PubMed PMC

Wenzl P, Suchánková P, Carling J, Simková H, Huttner E, Kubaláková M, Sourdille P, Paul E, Feuillet C, Kilian A, et al. (2010) Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps. Theor Appl Genet 121: 465–474 PubMed

Wicker T, Mayer KFX, Gundlach H, Martis M, Steuernagel B, Scholz U, Šimková H, Kubaláková M, Choulet F, Taudien S, et al. (2011) Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives. Plant Cell 23: 1706–1718 PubMed PMC

Zhou RN, Hu ZM. (2007) The development of chromosome microdissection and microcloning technique and its applications in genomic research. Curr Genomics 8: 67–72 PubMed PMC

Flow Cytometric Analysis and Sorting of Plant Chromosomes

Chromosome analysis and sorting

Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses

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

Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping

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JX624129, JX624130, JX624131, JX624132, JX624133, JX624134, JX624135, JX624136