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Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo‐intraspecific variation in genome size. Two important compounds present in coffee trees—caffeine and chlorogenic acid—modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes.

The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4¢,6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C-PI or C-DAPI) was compared with that of the standard, petunia (P-PI or P-DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R-PI or R-DAPI) is expected to be proportional to the genome size. Between-tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between-tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between-tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed. a 2002 Annals of Botany Company

Discrepancies of 2–20% were noted in nuclear DNA content estimates using mixed and unmixed extracts of several plant species combinations (target/standard). This may be because cytosolic components affect dye accessibility to DNA. Several dilution experiments showed clearly how cytosol concentration modified dye accessibility. Heat treatment suggested that dye accessibility could be related to chromatin sensitivity to decondensation. Some methods (nucleus isolation, dilution) can decrease, but not eliminate, stoichiometric error. The high sensitivity of the nucleus to the medium highlights the problem of reliability limits in estimating genome size. The choice between internal and external standardization and the interpretation of intraspecific variation in ‘nuclear DNA content’ are discussed.

Cassava (Manihot esculenta Crantz) cDNA clones were used to detect restriction fragment length polymorphisms in a collection of Manihot germplasm maintained as in vitro plants at ORSTOM, Montpellier. The collection consisted of mostly African cultivars of M. esculenta, together with a few M. glaziovii Mueller von Argau and M. caerulescens Pohl, and some interspecific hybrids between M. esculenta and M. glaziovii. The clones revealed significant levels of polymorphism both within and between the species; sufficient to construct dendrograms indicating the genetic diversity within the collection.

• 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 °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 °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 °C and 70 °C. Consequences are discussed and stoichiometric error detection methods are proposed, along with proposals for minimizing them.

Flow cytometry was conducted to evaluate genome size diversity among African diploid species of the Coffea genus. The study included 15 species and six new taxa from Congolese and Cameroonian forest regions which have yet to be botanically characterized. Between‐population differences were also recorded in some cases. These evaluations using an internal standard were highly correlated with previous results obtained with an external standard, but differences of up to 18 % existed for some species, involving stoichiometric errors. Consequently, genome size variation between species and within species are discussed as true genome size differences or stoichiometric errors. Environmental and phenotypic correlations with genome size are also discussed.