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Flow cytometric chromosome sorting in plants: The next generation
Jan Vrána, Hana Šimková, Marie Kubaláková, Jarmila Číhalíková, Jaroslav DoleŽel
a b s t r a c t: Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.
Conclusion: This protocol describes a procedure for preparation of plant chromosome DNA for next-generation sequencing. To date, it has been used in barley, rye and wheat. However, it can be adapted to other species. For example, it was used recently to sequence chromosomes from fescue and goatgrasses ( Kopecký et al., in preparation). The modifications concern the procedure for preparation of liquid chromosome suspensions (synchronization of the cell cycle in root tip meristems and release of chromosomes into solution).
Acknowledgements: This work was supported by the Czech Science Foundation (award No. P501/10/1740) and by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01).
