Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.
We argue that there is a need to maintain the term genome size in a broad sense as a covering term, because it is widely understood, short and phonetically pleasing. Proposals are made for a unified and consensual terminology. In this, 'genome size' should mean the DNA content based on chromosome number x and n, and should be used mainly in a general sense. The necessary distinction of the kinds of genome sizes is made by the adjectives 'monoploid' and the neology 'holoploid'. 'Holoploid genome size' is a shortcut for the DNA content of the whole chromosome complement characteristic for the individual (and by generalization for the population, species, etc.) irrespective of the degree of generative polyploidy, aneuploidies, etc. This term was lacking in the terminology and is for reasons of linguistic consistency indispensable. The abbreviated terms for monoploid and holoploid genome size are, respectively, Cx-value and C-value. Quantitative data on genome size should always indicate the C-level by a numerical prefix, such as 1C, 1Cx, 2C, etc. The proposed conventions cover general fundamental aspects relating to genome size in plants and animals, but do not treat in detail cytogenetic particularities (e.g. haploids, hybrids, etc.) which will need minor extensions of the present scheme in a future paper.
Three independent databases of eukaryotic genome size information have been launched or re-released in updated form since 2005: the Plant DNA C-values Database (), the Animal Genome Size Database () and the Fungal Genome Size Database (). In total, these databases provide freely accessible genome size data for >10 000 species of eukaryotes assembled from more than 50 years' worth of literature. Such data are of significant importance to the genomics and broader scientific community as fundamental features of genome structure, for genomics-based comparative biodiversity studies, and as direct estimators of the cost of complete sequencing programs.
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