BackgroundDespite the fact that coffee rust was first investigated scientifically more than a century ago, and that the disease is one of the major constraints to coffee production - constantly changing the socio-economic and historical landscape of the crop - critical aspects of the life cycle of the pathogen, Hemileia vastatrix, remain unclear. The asexual urediniospores are regarded as the only functional propagule: theoretically, making H. vastatrix a clonal species. However, the well-documented emergence of new rust pathotypes and the breakdown in genetic resistance of coffee cultivars, present a paradox.Methods and ResultsHere, using computer-assisted DNA image cytometry, following a modified nuclear stoichiometric staining technique with Feulgen, we show that meiosis occurs within the urediniospores. Stages of spore development were categorised based on morphology, from the spore-mother cell through to the germinating spore, and the relative nuclear DNA content was quantified statistically at each stage.ConclusionsHidden sexual reproduction disguised within the asexual spore (cryptosexuality) could explain why new physiological races have arisen so often and so quickly in Hemileia vastatrix. This could have considerable implications for coffee breeding strategies and may be a common event in rust fungi, especially in related genera occupying the same basal phylogenetic lineages.

The oleaginous species Acrocomia aculeata produces high-quality oil and is considered a potential plant for sustainable production of food and biodiesel. In spite of its economical, social and environmental importance, few data concerning the genome size and chromosomal characterisation of this crop have been reported. In order to contribute to basic genetic knowledge on A. aculeata, this work aimed to assemble the first karyogram and to determine genome size and base composition of this species. Concerning the cytogenetic approach, we developed a protocol based on root tips treatment with an anti-mitotic agent, followed by enzymatic maceration and slide preparation by the air-drying technique. This method provided well resolved metaphasic chromosomes, which are important for an accurate and informative cytogenetical characterisation. A chromosome number of 2n = 30 was observed. Content of 2C DNA and base composition were estimated by flow cytometry of G0/G1 nuclei stained with propidium iodide and 4′,6-diamidino-2-phenylindole, respectively. The mean 2C-value and base composition corresponded to 2C = 5.81 pg and AT = 58.3%. These new data support basic genetic knowledge on A. aculeata, relevant for its conservation, diversity studies and consequent development of breeding programs, which may foment the biofuel production in the world.

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The genome size of most rust species is unknown due, in part, to technical constraints, especially the difficulty in accessing spores to extract the nuclei for cytometry. Using the urediniospores of coffee rust, Hemileia vastatrix, an improved methodology involving flow cytometry was developed for accurate measurement of the nuclear genome size. The results revealed that the genome of this primitive rust fungus is unusually large -measuring 1C-value = 0Á75 pg (733Á5 Mb) -significantly bigger than other species quantified thus far in the more advanced rust lineages. The evolutionary consequences and the potential ecological constraints of this large genome size are discussed in relation to the epidemiology of coffee rust.

The genus Eucalyptus L’Herit. is the most cytologically studied group of plants in the family Myrtaceae. Despite the ecological and economic importance of this genus, much of its biology remains poorly understood. Cytological methods are useful in evolutionary studies; however, karyomorphological descriptions of the Eucalyptus species are limited. In this study, we developed a cytogenetic methodology that facilitates karyogram assembly and chromosome classification of Eucalyptus citriodora. The protocol includes treatment of root tips with amiprophos-methyl in Hoagland’s solution, slide preparation by cell dissociation, and an air-drying technique. Using this method, well-resolved metaphasic chromosomes with different compaction levels were obtained. A chromosome number of 2n = 22 was confirmed. On the basis of metaphasic chromosomes with limited condensation, E. citriodora showed six metacentric and four submetacentric chromosomes, and one submetacentric chromosome with a nucleolus organizer region (NOR). Thus, the informative chromosomes obtained in this study has enabled the most precise karyotype description of a Eucalyptus species, to date.