Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation

Abstract

Background and aims: In microdensitometry and flow cytometry, estimation of nuclear DNA content in a sample requires a standard with a known nuclear DNA content. It is assumed that dye accessibility to DNA is the same in the sample and standard nuclei. Stoichiometric error arises when dye accessibility is not proportional between the sample and standard. The aim of the present study was to compare the effects of standardization (external-internal) on nuclear fluorescence of two Coffea species and petunia when temperature increases, and the consequences on genome size estimation.

Methods: Two coffee tree taxa, C. liberica subsp dewevrei (DEW) and C. pseudozanguebarieae (PSE), and Petunia hybrida were grown in a glasshouse in Montpellier, France. Nuclei were extracted by leaf chopping and at least 2 h after nuclei extraction they were stained with propidium iodide for approx. 3 min just before cytometer processing. In the first experiment, effects of heat treatment were observed in mixed (DEW + petunia) and unmixed extracts (petunia and DEW in separate extracts). Nine temperature treatments were carried out (21, 45, 55, 60, 65, 70, 75, 80 and 85 degrees C). In a second experiment, effects of heating on within-species genome size variations were investigated in DEW and PSE. Two temperatures (21 and 70 degrees C) were selected as representative of the maximal range of chromatin decondensation.

Key results and conclusions: In coffee trees, sample and standard nuclei reacted differently to temperature according to the type of standardization (pseudo-internal vs. external). Cytosolic compounds released in the filtrate would modify chromatin sensitivity to decondensation. Consequently, the 'genome size' estimate depended on the temperature. Similarly, intraspecific variations in genome size changed between estimations at 21 degrees C and 70 degrees C. Consequences are discussed and stoichiometric error detection methods are proposed, along with proposals for minimizing them.

Fig. 1.

Within-tree relationships comparing the petunia peak location and the coffee peak location. Each symbol represents one tree (from Noirot et al. 2002).

Fig. 2.

Effects of temperature on petunia nuclear fluorescence in external standardization [without Coffea liberica subsp. dewevrei (DEW)] and pseudo-internal standardization (with DEW). Fluorescence is expressed in channel units and was recorded using propidium iodide.

Fig. 3.

Effects of temperature on Coffea liberica subsp. dewevrei (DEW) nuclear fluorescence in external standardization (without petunia) and pseudo-internal standardization (with petunia) Fluorescence is expressed in channel units and was recorded using propidium iodide.

Fig. 4.

Effects of temperature on the peak fluorescence ratio in external standardization (unmixed filtrate) and pseudo-internal standardization (mixed filtrate).

Fig. 5.

Effects of temperature (21–85 °C; see numbers on the points) on the relationship between Coffea liberica subsp. dewevrei (DEW) and petunia nuclear fluorescence in (A) pseudo-internal standardization conditions; and (B) external standardization conditions. The regression line computed in (A) is also reproduced in (B). Note that the scales are identical in (A) and (B) to highlight differences.

Fig. 6.

Relationship between slopes and intersects for within-tree linear regressions intersecting at one point (450·3; 894·8).