Recalibration of opaque-2 line of maize as a standard to estimate nuclear DNA amount
--Rosato, M; Naranjo, CA; Poggio, L

Measurements of DNA amount by microdensitometry usually are based on nuclei stained by the Feulgen method. It is generally accepted that Feulgen staining is specific for DNA (after removal of RNA by acid hydrolysis) and its proportionality between stain density and DNA amount can be assumed. Consequently the light absorption of a nucleus so stained is a quantitative measure of its DNA content (Bennett and Smith, Proc. Roy. Soc. London B:227-274, 1996).

When nuclear DNA content is measured by this method the results are obtained in arbitrary units (A.U.). The arbitrary units can be converted into absolute units in picograms (pg), through a standard species the DNA amount of which is already known.

Bennett and Smith (l.c.) gave the criteria to recalibrate several species against Allium cepa cv. Ailsa Craig (2C= 33.5 pg) in order to justify their use as known standards for calibrating other species. This procedure tends to minimize some technical errors inherent in Feulgen microdensitometry.

The standards available for species of the genus Zea in the literature are 2C= 5.155 pg for Va35 line (Rayburn et al., Am. J. Bot. 72:1610-1617, 1985; Rayburn et al., J. Exp. Bot. 40:1179-1183, 1989), and 5.9 pg for c-tester line (Tito, Poggio and Naranjo, MNL 65:76, 1991). The latter is cultivated in the IFSC (FCAF, UNLP) and has reduced vigor and fertility as a result of many generations of inbreeding. Because in our laboratory we are working in intra- and interpopulation variation of DNA amount of Argentine races of maize, it was convenient to recalibrate a line available in our country and with good fertility.

The opaque-2 line (raised by M. Aulicino, IFSC) has all the characteristics suggested by Bennett and Smith to justify its use as standard; therefore, four experiments were done on different days to recalibrate carefully the 2C value of opaque-2 line against Allium cepa cv. Ailsa Craig. The technique of staining was performed as described by Tito, Poggio and Naranjo (Theor. Appl. Genet. 83:58-64,1991).

DNA content was measured in 20 telophase nuclei (2C) of the root tips of germinating seeds. Roots of 0.5-1 cm in length were fixed in 3:1 (ethanol:acetic acid). After fixation, the roots were rinsed for 30 min in distilled water. Hydrolysis was carried out with 5 N HCl at 20 C for 30 min The roots were then washed three times in distilled water for 15 min, and stained for 120 min in Schiff's reagents at pH 2.2. The material was then rinsed three times in SO2 water for 10 min each, kept in distilled water and squashed in 45% acetic acid. The coverslip was removed after freezing with CO2 and the slide was dehydrated in absolute alcohol and then mounted in Euparal. The amount of Feulgen staining per nucleus, expressed in arbitrary units, was measured at a wavelength of 570 nm, using the scanning method with a Zeiss Universal Microspectrophotometer (UMSP 30). The DNA content per basic genome expressed in pg was calculated using A. cepa cv. Ailsa Craig as a standard. The differences in DNA content were tested through an analysis of variances and comparisons between means using Scheffe's method. The results obtained are shown in Table 1.

An anova test was made, and it was determined that the results do not show significant differences among themselves (F= 1.7203, p< 0.05). The results obtained point out a DNA average amount for opaque-2 line 2C= 6.658 ±0.038 pg.

Table 1. Recalibration of opaque-2 line as a standard to estimate nuclear DNA amount.
 
Experiment
Individual Nuclei measured (No.) DNA content ±SE (A.U.) (2C) ±SE (pg)
1 1 20 13.34 ± 0.14 6.678 ± 0.07
2 20 13.32 ± 0.17 6.662 ± 0.08
3 20 13.29 ± 0.11 6.620 ± 0.06
2 4 18 13.60 ± 0.13 6.799 ± 0.06
19 15.60 ± 0.16 6.736 ± 0.06
6 10 15.81 ± 0.34  6.800 ± 0.15
3 7 20 15.82 ± 0.14  6.644 ± 0.06
8 15 14.96 ±0.19 6.738 ± 0.08
4 9 20 13.17 ± 0.19 6.454 ± 0.09
10 10 13.05 ± 0.33 6.450 ± 0.15


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