Nuclear genome size: Are we getting closer?

Abstract

Correct information on genome size is important in many areas of research. For a long time, scientists have been struggling to understand the reason for the huge variation in eukaryotic genome size and its biological significance. More recently, the knowledge on genome size has become important to structure genome sequencing projects as their scale and cost depend on genome size. Despite the fact that the first estimates of genome size in eukaryotes were made more than 50 years ago, we are still not quite sure about the exact genome size in practically all animal and plant species. Moreover, different estimates continue to be published for the same species. These discrepancies compromise data comparison and interpretation and point to methodological problems, which include standardization. This article assesses the current state of DNA reference standards for flow cytometry and the issues related to their calibration. ' 2010 International Society for Advancement of CytometryKey terms cytometric techniques; reference standards; genome sequencing; conversion factor; nuclear DNA content; C-value MOST of the genetic information in eukaryotes is localized in the cell nucleus and numerous attempts have been made to determine the quantity of nuclear DNA, especially after various lines of evidence associated DNA with genes (1). It soon became clear that the amount of DNA per nucleus is relatively constant in somatic cells of a given species and that sperm cells have approximately half the DNA amount found for somatic cells (2,3). These experiments provided independent data to support the hereditary role of DNA and marked the beginning of numerous fruitful lines of research and applications, many of them still important today, all relying on the ability to determine DNA amounts in cell nuclei.The early determinations were made colorimetrically on DNA extracted from a known number of cells although the presence of cells in different cell cycle phases could compromise the accuracy of these estimates. The colorimetric approach made identification of subpopulations of cells with different DNA amounts impossible. However, cytometric methods suitable for measurement of absorbance of light in individual nuclei (either using UV light with non-stained nuclei and/or monochromatic visible light with nuclei stained by the Feulgen reaction) were already available during the 1940s and were used to discover the presence of cells with different classes of DNA amounts. Such cells were observed in a variety of animal tissues and were associated with polyploidy and polyteny (4,5). The presence of nuclei with 2, 4, 8, 16, or 32 times the haploid value was described in plant tissues by Swift (6) who introduced the ''C'' terminology to classify nuclear DNA amounts. In this terminology, DNA classes are labeled as C, 2C, 4C, 8C, etc. to characterize DNA amounts of nuclei by multiples of the DNA amount in a complete chromosome set in a non-replicated haploid nucleus, which has the class C DNA amount.At the beginning of the 1950s, biochem...

New reports of nuclear DNA content for 66 traditional Chinese medicinal plant taxa in China

“…Nevertheless, flow cytometry is not without its problems and careful application of the technique is essential for generating reliable data (Doležel et al 2007a;Greilhuber et al 2007). Indeed, there are multiple factors that can affect the reliability of C-value data, including the choice of extraction buffer (with over 28 different buffers in use; Lourerio et al 2007), how raw FCM data are analyzed (forward scatter, side scatter and relative fluorescence intensity), which plant species are used as calibration standards (Doležel and Greilhuber 2010;Suda and Leitch 2010) and the potentially confounding effects of cytosolic compounds (e.g. anthocyanin and tannic acid) (Doležel et al 2007a(Doležel et al , 2007bBennett and Leitch 2011;Loureiro, Suda, et al 2007;Loureiro, Rodriguez, et al 2007;Greilhuber et al 2007).…”

Flow Cytometry Applied in Tissue Culture

“…This pattern receives two reference designations internal standard, when extraction of the cores and the analysis of sample and standard are performed simultaneously, or when an external standard is performed separately. The internal standards are most recommended, especially in high-precision measurements, because the peaks of the standard used and the sample appear in the same histogram and are treated under identical conditions [26] thereby reducing possible errors due to oscillation of the device during the evaluation of the samples. However [27] reported that the simultaneous processing of the sample and the reference standard was not necessary to obtain reliable estimates of DNA.…”

DNA content estimation of Fig and Black Mulberry using flow cytometry

“…Feulgen densitometry (FD) and flow cytometry (FC) are the two mostly used methods in DNA content estimation. The latter has been used to estimate the nuclear DNA content and genome size of an unknown sample comparing with a reference standard (GALBRAITH et al, 1983;DOLEŽEL et al, 1998;GREILHUBER, 2010;TİRYAKİ;TUNA, 2012). Different fluorochromes (fluorescent dyes) are used in FC.…”