Flow sorting and sequencing meadow fescue chromosome 4F

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  • David Kopecký, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Mihaela Martis, Munich Information Center for Protein Sequences/Institute of Bioinformatics and Systems Biology, Germany
  • Jarmila Číhalíková, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Eva Hřibová, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Jan Vrána, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Jan Bartoš, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Jitka Kopecká, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Federica Cattonaro, Istituto di Genomica Applicata and Istituto di Genomica Applicata Technology Services, Italy
  • Štěpán Stočes, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Petr Novák, Biology Centre Academy of Sciences of the Czech Republic, Institute of Plant Molecular Biology, Czech Republic
  • Pavel Neumann, Biology Centre Academy of Sciences of the Czech Republic, Institute of Plant Molecular Biology, Czech Republic
  • Jiří Macas, Biology Centre Academy of Sciences of the Czech Republic, Institute of Plant Molecular Biology, Czech Republic
  • Hana Šimková, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Bruno Studer, Institute for Agricultural Sciences, Switzerland
  • Torben Asp
  • James H Baird, Department of Botany and Plant Sciences, University of California, United States
  • Petr Navrátil, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Miroslava Karafiátová, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Marie Kubaláková, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Jan Šafář, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
  • Klaus Mayer, Munich Information Center for Protein Sequences/Institute of Bioinformatics and Systems Biology, Germany
  • Jaroslav Doležel, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Czech Republic
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.
Original languageEnglish
JournalPlant Physiology
Volume163
Issue3
Pages (from-to)1323-1237
Number of pages15
ISSN0032-0889
DOIs
Publication statusPublished - Nov 2013

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