A flow cytometry protocol for accurate and precise measurement of plant genome size using frozen material

Abstract

AbstractFlow cytometry is a technique widely applied to infer the ploidy and genome size of plant nuclei. The conventional approach of sample preparation, reliant on fresh plant material to release intact nuclei, requires protocol optimisation for application to many species. The approach often results in poor yields of nuclei, impeding the accurate measurement of genome size and confines the optimal resource allocation and efficiency in genome sequencing which relies on genome size estimation. Here, we present a novel method using frozen plant material that facilitates the release of intact nuclei for genome size estimation. Genome estimates from frozen material are similar to those from fresh material. Accurate and precise estimates can be made by complementing the fluorescence of frozen nuclei with histogram modelling and debris compensation algorithms. This method of nuclei isolation from frozen plant material for flow cytometry-based genome size estimations has special value in estimating the genome size of samples collected and frozen for use in plant genome sequencing. Plant material can be conveniently stored, resampled, and used for DNA or RNA extractions.HighlightFrozen leaf material can be used to isolate nuclei for the accurate estimation of genome size The method proved suitable for difficult samples and did not require specific optimization. The method was especially useful where plant material could not be immediately processed through flow cytometry and allowed the same sample to be used for genomes size estimation and genome sequencing.