Reliability of CValues

A reliable C-value should be established, optimally based on measurements by different laboratories. This is a sensitive point, because in fact only one C-value for a plant standard evaluated using a method yielding absolute amounts of DNA is generally accepted. This is Allium cepa (Alliaceae), whose nuclear DNA content per root tip meristem cell (expectedly corresponding to roughly 3C) has been chemically determined as 54.3 pg by Sparrow and Miksche (1961) and was re-calculated as 2C = 33.55 pg by Van't Hof (1965) who took into account the relative lengths of the mitotic cycle phases. This value agrees well with chemically determined values obtained from animals and humans using the Feulgen cyto-photometric comparison (Greilhuber et al. 1983). Almost all other trustworthy C-values for plants are based on cytometric comparisons with plants and lastly with onion, or with human and animals, for which chemical estimates exist. The old chemical estimates in the human vary between 1C = 3.0 and 3.5 pg (Metais et al. 1951; Vendrely and Vendrely 1949). Many authors arbitrarily used the higher value for their calibrations, although a value of 3.1-3.2 pg may be closer to the truth (Dolezel et al. 2003; Greilhuber et al. 1983). In one important recent investigation (Bennett et al. 2003), the size of a completely sequenced genome size was already known, that is, of the nematode Caenorhabditis elegans, which was used for FCM

86 4 Nuclear DNA Content Measurement 400

Fig. 4.3 Simultaneously prepared and measured propidium-iodide stained nuclear suspensions of Arab idopsis thaliana ''Columbia'' and chicken (a) and Caenorhabditis elegans ''Bristol N2'' (b), respectively. The positions of chicken 2C relative to A. thaliana 16C and of C. elegans 4C versus A. thaliana 2C give an indication of the genome size of A. thaliana and chicken on the basis of a C. elegans lC-value of 100 Mbp. For details see text. (From Bennett et al. 2003 with permission).

Fig. 4.3 Simultaneously prepared and measured propidium-iodide stained nuclear suspensions of Arab idopsis thaliana ''Columbia'' and chicken (a) and Caenorhabditis elegans ''Bristol N2'' (b), respectively. The positions of chicken 2C relative to A. thaliana 16C and of C. elegans 4C versus A. thaliana 2C give an indication of the genome size of A. thaliana and chicken on the basis of a C. elegans lC-value of 100 Mbp. For details see text. (From Bennett et al. 2003 with permission).

comparison with Arabidopsis thaliana ''Columbia''. For this important plant species, a value of 1C = 157 Mbp was estimated using FCM, based on 1C = 100 Mbp for this worm (Fig. 4.3). This example clearly showed the fragility of the value of 125 Mbp published by the Arabidopsis Genome Initiative (2000), which significantly underestimated the non-sequenced DNA harbored in the heterochromatin (Bennett et al. 2003). But note that A. thaliana collected in the wild was meanwhile reported to vary by about 10% in genome size between accessions (Schmuths et al. 2004). There is clearly a need for in-depth analyses of genome sizes of plant standard species to arrive at agreed absolute values.

Studies on Plant Standards

DoleZel et al. (1998) were the first to compare a set of nine different standard species of defined cultivars or lines in four laboratories with PI and also with DAPI, and laser and lamp-based flow cytometers, and with Feulgen scanning densitometry. The species were compared in a cascade-like manner starting from Allium cepa (assumed to be 2C = 33.55 pg) down to Arabidopsis thaliana, with a mean result of 2C = 0.37 pg, while 0.321 pg is the expected value reported by Bennett

Table 4.5 Ratios of C-values and relative standard deviations (N = 10) estimated for pairs of species by four laboratories (L1-L4). Nuclei were isolated simultaneously and stained with propidium iodide. A.c. Allium cepa, V.f. Viciafaba, S.c. Secale cereale, H.v. Hordeum vulgare, P.s. Pisum sativum, Z.m. Zea mays, G.m. Glycine max, R.s. Raphanus sativus, A.t. Arabidopsis thaliana. (Adapted from Dolezel et al. 1998).

V.f./ S.c./ H.v./ P.s./ Z.m./ G.m./ R.s./ A.t./ A.c. V.f.S.c. H.v. P.s. Z.m. G.m. R.s.

Lllal

0.778

0.613

0.647

0.874

0.639

0.469

0.506

0.310

(0.9)

(1.0)

(0.6)

(1.0)

(3.3)

(6.6)

(1.2)

(1.0)

L4lal

0.792

0.606

0.661

0.869

0.658

0.519

0.464

0.302

(3.5)

(2.8)

(0.8)

(0.9)

(2.9)

(0.8)

(0.6)

(0.3)

L2[b

0.776

0.595

0.638

0.863

0.609

0.441

0.462

0.300

(1.3)

(0.8)

(0.8)

(0.8)

(1.3)

(1.6)

(1.7)

(0.7)

L3[b

0.752

0.586

0.632

0.879

0.586

0.438

0.465

0.313

(2.1)

(1.4)

(0.8)

(0.5)

(0.5)

(0.8)

(1.9)

(3.5)

Mean ratio

0.774

0.600

0.645

0.870

0.623

0.467

0.474

0.306

Largest difference between

1.8

1.1

2.1

0.6

2.9

9.6

8.3

2.6

laser cytometers (%)

Largest difference between

3.1

1.5

0.9

1.8

3.8

0.7

0.6

4.2

lamp cytometers (%)

Largest difference (all

5.1

4.4

4.4

1.8

10.9

15.6

8.7

a Laser-based instruments. b Lamp-based instruments.

et al. (2003). Feulgen DNA measurements with 2C = 0.326 pg closely approached this value. The four laboratories produced strongly correlated data although the types of cytometer used differed in that laser instruments seemed to slightly underestimate the larger genomes. Nevertheless, some critical differences between laboratories were noticed (Table 4.5). Ratios of single species pairs differed by up to 15.9% (mean 6.9%), which was higher than anticipated. Laser instruments produced results which differed by up to 9.6% (mean 3.6%), and with lamp-based instruments the results differed by up to 3.8% (mean 2.1%; Table 4.5). These differences are difficult to explain but may be related to instrument-specific linearity bias, differences in the growth conditions of the plants, the use of different plant parts and perhaps also to the use of different buffers, which according to Loureiro et al. (2006a) can influence the various species investigated somewhat differently (see above).

Johnston et al. (1999) conducted a study on plant standards for FCM involving two laboratories, using among other crop species Pisum sativum ''Minerva Maple'', Hordeum vulgare ''Sultan'' (Poaceae), Vicia faba ''GS011'', and Allium cepa ''Ailsa Craig''. This study revealed problems with CRBC variability, and compared with Dolezel et al. (1998) generally yielded somewhat higher 2C-values for P. sativum (9.56 vs. 8.75 pg), H. vulgare (11.12 vs. 10.04 pg), and V.faba (26.66 vs. 25.95 pg). The value for Allium cepa was accepted to be 33.55 pg. As already mentioned, in this study, the beetle Tetraodes sp. (2C = 1.0 pg) was the primary standard; it served for two chicken accessions whose 2C-values were quite different i.e. 2.49 and 3.01 pg. Of these, the higher (and probably too high) value of 3.01 pg was used for calibrating H. vulgare, which was then used to calibrate the remaining species (Johnston et al. 1999). It seems that assuming a too high value for the chicken is the main reason for the higher plant DNA values given by Johnston et al. (1999) compared to Dolezel et al. (1998).

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