Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

• Keywords
• Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
• MeSH
• Chromosomes, Plant * genetics   MeSH
• Genome, Plant MeSH
• Flow Cytometry methods   MeSH
• Restriction Mapping MeSH
• Plants * genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

• Keywords
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• Chromosomes, Plant * MeSH
• Chromosomes * MeSH
• Cytogenetics MeSH
• Karyotyping MeSH
• Flow Cytometry methods   MeSH
• Suspensions MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Suspensions MeSH