Methods used to estimate genome size are reviewed in Peterson (2014). The
easiest and most widely accepted method of obtaining a genome size estimate is
through the use of flow cytometry. In brief, isolated nuclei in solution are stained
with a fluorescent quantitative DNA stain (most commonly, propidium iodide). The
stained nuclei are gently pulled into a thin stream of liquid whose diameter is so
narrow that the fluid exhibits almost laminar flow. The nuclei move into the stream
one at a time; if multiple nuclei try and enter the stream, the flow is disrupted. The
liquid stream passes through a laser which excites the fluorescent DNA stain in the
nuclei. As it passes through the laser, each nucleus gives off a burst of fluorescence
that is proportional to the amount of DNA it carries. Measurements from thousands
of nuclei can be obtained in just a few minutes. Stained nuclei from an organism of
known genome size are usually included as an internal standard facilitating accurate
genome size estimation.
One might think that whole genome sequencing would provide the most accurate
genome size determinations. However, plant genome assemblies typically contain
gaps of uncertain size. Such gaps are especially prevalent at centromeres, telomeres,
and in other highly repetitive genome regions. Based on the Arabidopsis thaliana
genome assembly (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001735.3;
accessed on March 2, 2018), the genome size of A. thaliana would be predicted to
be 119.9 Mb (119.7 Mb in sequence assemblies plus 0.2 Mb in gaps). However, in a
study led by Michael Bennett, the world’s foremost expert on plant genome sizes
(Bennett et al. 2003), the authors convincingly showed that the Arabidopsis thaliana
genome is about 157 Mb, a value that agrees with previous Feulgen densitometry
and flow cytometry measurements. Thus the first and arguably best plant genome
assembly is probably only 76% complete. Until considerable advances are made in
sequencing and assembly of genomes, Feulgen densitometry and FCM should be
viewed as the most accurate means of determining genome sizes (Peterson 2014).
2.5 C-Value Paradox/Enigma
In 1944, it was shown that DNA is the molecule in which genetic information is
stored (Avery et al. 1944). In 1948, it was revealed that species had relatively stable
genome sizes that could be measured (Boivin et al. 1948), and in 1950 the term 1C
DNA value was coined (Swift 1950). By 1951, scientists already had acquired
enough genome size estimates to notice that 1C DNA contents of eukaryotic
organisms did not seem to be correlated with organismal structural complexity.
4
Mirsky and Ris (1951) deemed this phenomenon the “C-value paradox.” The
assumption at the time was that since all genes were encoded by DNA, then all
4 For example, the lily, Paris japonica, does not need or have 955 times as many genes as
Arabidopsis thaliana, although the two species have a 955-fold difference in 1C DNA content
(Pellicer et al. 2010; Bennett et al. 2003).
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D. G. Peterson and M. Arick
easiest and most widely accepted method of obtaining a genome size estimate is
through the use of flow cytometry. In brief, isolated nuclei in solution are stained
with a fluorescent quantitative DNA stain (most commonly, propidium iodide). The
stained nuclei are gently pulled into a thin stream of liquid whose diameter is so
narrow that the fluid exhibits almost laminar flow. The nuclei move into the stream
one at a time; if multiple nuclei try and enter the stream, the flow is disrupted. The
liquid stream passes through a laser which excites the fluorescent DNA stain in the
nuclei. As it passes through the laser, each nucleus gives off a burst of fluorescence
that is proportional to the amount of DNA it carries. Measurements from thousands
of nuclei can be obtained in just a few minutes. Stained nuclei from an organism of
known genome size are usually included as an internal standard facilitating accurate
genome size estimation.
One might think that whole genome sequencing would provide the most accurate
genome size determinations. However, plant genome assemblies typically contain
gaps of uncertain size. Such gaps are especially prevalent at centromeres, telomeres,
and in other highly repetitive genome regions. Based on the Arabidopsis thaliana
genome assembly (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001735.3;
accessed on March 2, 2018), the genome size of A. thaliana would be predicted to
be 119.9 Mb (119.7 Mb in sequence assemblies plus 0.2 Mb in gaps). However, in a
study led by Michael Bennett, the world’s foremost expert on plant genome sizes
(Bennett et al. 2003), the authors convincingly showed that the Arabidopsis thaliana
genome is about 157 Mb, a value that agrees with previous Feulgen densitometry
and flow cytometry measurements. Thus the first and arguably best plant genome
assembly is probably only 76% complete. Until considerable advances are made in
sequencing and assembly of genomes, Feulgen densitometry and FCM should be
viewed as the most accurate means of determining genome sizes (Peterson 2014).
2.5 C-Value Paradox/Enigma
In 1944, it was shown that DNA is the molecule in which genetic information is
stored (Avery et al. 1944). In 1948, it was revealed that species had relatively stable
genome sizes that could be measured (Boivin et al. 1948), and in 1950 the term 1C
DNA value was coined (Swift 1950). By 1951, scientists already had acquired
enough genome size estimates to notice that 1C DNA contents of eukaryotic
organisms did not seem to be correlated with organismal structural complexity.
4
Mirsky and Ris (1951) deemed this phenomenon the “C-value paradox.” The
assumption at the time was that since all genes were encoded by DNA, then all
4 For example, the lily, Paris japonica, does not need or have 955 times as many genes as
Arabidopsis thaliana, although the two species have a 955-fold difference in 1C DNA content
(Pellicer et al. 2010; Bennett et al. 2003).
118
D. G. Peterson and M. Arick