High-throughput monitoring of plant nuclear DNA contents via flow cytometry

Abstract

Interest in measuring the nuclear holoploid genome sizes of higher plants reflects not just the status of the nucleus as a defining characteristic of eukaryotic organisms. Higher plants also attract interest in that they display an unusually large range of genome sizes, current measurements indicating an almost 2,500-fold difference between the smallest and the largest. Scientists would like to learn more about the significance of nuclear genome sizes, in terms of molecular and cytological mechanisms regulating the interaction of the nucleus with the cytoplasm and regulating the observed increases and decreases of genome sizes observed within and across families, genera, and species. We would like to understand their adaptive significance through charting their distribution within populations and ecosystems. Further, since genome size values are only available for a small minority of the ∼650,000 species of angiosperms (known and yet undiscovered), we would like to systematically survey plant genome sizes globally before their extinction as a consequence of anthropogenic change. Flow cytometry is accepted as the method of choice for genome size measurements, these measurements being based on fluorescent staining of the nuclear DNA. Flow cytometry offers exceptional ease of use, accompanied by high accuracy and reproducibility and low cost. This chapter provides a general discussion of flow cytometric methods for measuring plant genome sizes, and detailed methods for carrying out these analyses.