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The Polyploid Gene Assembler (PGA), developed and tested in this study, represents a new strategy to perform gene-space assembly from complex genomes using low coverage DNA sequencing. The pipeline integrates reference-assisted loci and de novo assembly strategies to construct high-quality sequences focused on gene content. Pipeline validation was conducted with wheat (Triticum aestivum), a hexaploid species, using barley (Hordeum vulgare) as reference, that resulted in the identification of more than 90% of genes and several new genes. Moreover, PGA was used to assemble gene content in Saccharum spontaneum species, a parental lineage for hybrid sugarcane cultivars. Saccharum spontaneum gene sequence obtained was used to reference-guided transcriptome analysis of six different tissues. A total of 39,234 genes were identified, 60.4% clustered into known grass gene families. Thirty-seven gene families were expanded when compared with other grasses, three of them highlighted by the number of gene copies potentially involved in initial development and stress response. In addition, 3,108 promoters (many showing tissue specificity) were identified in this work. In summary, PGA can reconstruct high-quality gene sequences from polyploid genomes, as shown for wheat and S. spontaneum species, and it is more efficient than conventional genome assemblers using low coverage DNA sequencing.

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.

The eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts.

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 = 0·75 pg (733·5 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.

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.