| Autor: |
Gaiero P; Faculty of Agronomy, University of the Republic, Montevideo, Uruguay.; Laboratory of Genetics, Wageningen University & Research, Wageningen, The Netherlands., Šimková H; Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Olomouc, Czech Republic., Vrána J; Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Olomouc, Czech Republic., Santiñaque FF; Flow Cytometry and Cell Sorting Core, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay., López-Carro B; Flow Cytometry and Cell Sorting Core, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay., Folle GA; Flow Cytometry and Cell Sorting Core, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay., van de Belt J; Laboratory of Genetics, Wageningen University & Research, Wageningen, The Netherlands., Peters SA; Applied Bioinformatics, Department of Bioscience, Wageningen University & Research, Wageningen, The Netherlands., Doležel J; Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Olomouc, Czech Republic., de Jong H; Laboratory of Genetics, Wageningen University & Research, Wageningen, The Netherlands. |
| Abstrakt: |
Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies. |
