Genome-Wide Identification and Analysis of DOF Gene Family in Eugenia uniflora L. (Myrtaceae).

Eugenia uniflora is a Brazilian native plant species with great ecological and economic importance. It is distributed throughout the Atlantic forest, where two distinct populations show local adaptation to the contrasting conditions of restinga and riparian forest. Among various TFs described in plants, the DOF TF family has been reported to affect flowering and vascular development, making them promising candidates for characterization in E. uniflora. In this study, 28 DOF genes were identified by a genome-wide analysis, of which 20 were grouped into 11 MCOGs by Bayesian phylogeny, suggesting a shared functionallity between members. Based on RNA-seq experiments, we have detected eight drought responsive genes, and SNPs identification revealed population unique polymorphisms, implying a role in local adapatation mechanisms. Finally, analysis of conserved motifs through MEME revealed 15 different protein motifs, and a promoter region analysis returned 40 enriched TF binding motifs, both reporting novel biological functions circa the DOF gene family. In general, the DOF family is found to be conserved both in sequence and expression. Furthermore, this study contributes to both DOF literature and the genetic exploration of native species, elucidating their genetic potential and bringing to light new research topics, paving the way to future studies.

Chemical composition, antioxidant, antimicrobial activity, toxicity, genetic analysis and popular use of Eugenia luschnathiana (O. Berg) Klotzsch ex B. D. Jacks: a literature review / Composición química, actividad antioxidante, antimicrobiana, toxicidad, análisis genético y uso popular de Eugenia luschnathiana (O. Berg) Klotzsch ex B. D Jack: una revisión de la literatura

This review describes the geographical distribution, botanical data, popular use, chemical composition, pharmacological activities and genetic aspects related to Eugenia luschnathiana, a native Brazilian plant popularly known as "bay pitomba". E. luschnathiana leaves are characterized morphologically by the presence of a petiole, an attenuated base, acuminated apex, elliptical shape, and parallel venation. The major chemical compounds found in E. luschnathiana are sesquiterpenes. Literature reports showed that E. luschnathiana extracts have antioxidant properties and antimicrobial activity against Gram-negative and Gram-positive bacteria. The extractsfrom the leaf, fruit and stem, and a concentrated residual solution of its essential oil, displayed negligible toxicity. Lastly, a cytogenetic analysis indicated that some markers can be used for the study of genetic diversity, population structure, and genetic improvements. The information available on E. luschnathiana supports the hypothesis that this plant may be a source of compounds with promising pharmacological activity.

Esta revisión describe la distribución geográfica, datos botánicos, uso popular, composición química, actividad farmacológica y el análisis genético de Eugenia luschnathiana, una planta originaria del Brasil conocida popularmente como "pitomba da baía". Las hojas de E. luschnathiana se caracterizan por la presencia de pecíolo, base atenuada, ápice acuminado, forma elíptica y venación paralela. Su composición química presenta mayormente sesquiterpenos. Los informes en la literatura muestran que los extractos de E. luschnathiana presentan propiedades antioxidantes y actividad antimicrobiana contra las bacterias Gram-negativas y Gram-positivas. Los extractos de la hoja, fruto y tallo, y una solución residual concentrada del aceite esencial, presentaron baja toxicidad. Por último, un análisis citogenético indicó que algunos marcadores pueden utilizarse para estudios de diversidad genética, estructura poblacional y mejoramiento genético. Las informaciones disponibles acerca de E. luschnathiana proponen la hipótesis de que esta planta puede ser una fuente de compuestos con actividad farmacológica prometedora.

Chemical variability of essential oils of Eugenia uniflora L. genotypes and their antioxidant activity.

Eugenia uniflora, known as the "Brazilian cherry", is an economically important neotropical Myrtaceae in the cosmetics and pharmaceutical industries due the production of essential oils with antioxidant activity. On account of its significant genetic variability, genotype evaluations are needed in order to identify genetic features related to the essential oil production that meet the industry requirements. The main objective of the present study was to evaluate the yield, composition, and antioxidant activity of essential oils isolated from the leaves of 36 genotypes of E. uniflora. Essential oil samples were obtained by hydrodistillation, and their composition was determined by gas chromatography coupled with mass spectrometry. A variation of 0.22% to 1.68% in the essential oil yield was observed, in which 78 compounds, namely oxygenated sesquiterpenes, were identified. According to the cluster analysis of the major compounds, six groups were revealed. The observed diversity demonstrates the genetic variability of the species. Also, the antioxidant activity was affected by the composition of the essential oils, ranging from 176.66 to 867.57 µM TEAC.

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic factors is very relevant to help in understanding the processes of local adaptation. 1-Pyrroline-5-carboxylate synthetase (P5CS) is one interesting gene to study in this species since it encodes a key enzyme of proline biosynthesis, which is an osmoprotectant during abiotic stress. Applying in silico analysis, we identified one P5CS gene sequence of E. uniflora (EuniP5CS). Phylogenetic analysis, as well as, gene and protein structure investigation, revealed that EuniP5CS is a member of P5CS gene family. Plants of E. uniflora from two distinct environments (restinga and riparian forest) presented differences in the proline accumulation and P5CS expression levels under growth-controlled conditions. Both proline accumulation and gene expression level of EuniP5CS were higher in the genotypes from riparian forest than those from restinga. When these plants were submitted to drought stress, EuniP5CS gene was up-regulated in the plants from restinga, but not in those from riparian forest. These results demonstrated that EuniP5CS is involved in proline biosynthesis in this species and suggest that P5CS gene may be an interesting candidate gene in future studies to understand the processes of local adaptation in E. uniflora.

Discovery and characterization of SSR markers in Eugenia uniflora L. (Myrtaceae) using low coverage genome sequencing.

Eugenia uniflora L. (Myrtaceae) is a tree species widely distributed in South America suffering the effects of the exploitation of natural populations. In this study, we employed low coverage sequencing of the E. uniflora genome for mining of SSR markers. The de novo assembly generated 2,601 contigs with an average length of 1139 bp and spans 3.15 Mb. A total of 76 dimer, 33 trimer and two compound SSR loci were identified. Twelve selected SSR loci were employed to genotype 30 individuals from two natural populations. A total of 73 alleles were detected (mean A= 6.1) were observed, the mean effective number of alleles was Ae = 3.91, mean Ho was 0.23 and mean HE was 0.70). The mean Wright's within population fixation index was FIS = 0.66 and significant deviation of HWE was observed in all loci, except one. The FST between populations equaled 0.27. The levels of genetic diversity and structure estimated with these 12 SSR markers are in accordance with data from genetics studies performed on other tree species of the Pampa biome, presenting moderate to high polymorphism and may be employed in studies of species conservation measures and breeding programs.

Demographic stability and high historical connectivity explain the diversity of a savanna tree species in the Quaternary.

Background and Aims: Cyclic glaciations were frequent throughout the Quaternary and this affected species distribution and population differentiation worldwide. The present study reconstructed the demographic history and dispersal routes of Eugenia dysenterica lineages and investigated the effects of Quaternary climate change on its spatial pattern of genetic diversity. Methods: A total of 333 individuals were sampled from 23 populations and analysed by sequencing four regions of the chloroplast DNA and the internal transcribed spacer of the nuclear DNA. The analyses were performed using a multi-model inference approach based on ecological niche modelling and statistical phylogeography. Key Results: Coalescent simulation showed that population stability through time is the most likely scenario. The palaeodistribution dynamics predicted by the ecological niche models revealed that the species was potentially distributed across a large area, extending over Central-Western Brazil through the last glaciation. The lineages of E. dysenterica dispersed from Central Brazil towards populations at the northern, western and south-eastern regions. A historical refugium through time may have favoured lineage dispersal and the maintenance of genetic diversity. Conclusions: The results suggest that the central region of the Cerrado biome is probably the centre of distribution of E. dysenterica and that the spatial pattern of its genetic diversity may be the outcome of population stability throughout the Quaternary. The lower genetic diversity in populations in the south-eastern Cerrado biome is probably due to local climatic instability during the Quaternary.

The evolutionary history of Eugenia sect. Phyllocalyx (Myrtaceae) corroborates historically stable areas in the southern Atlantic forests.

BACKGROUND AND AIMS: Eugenia sect. Phyllocalyx Nied. includes 14 species endemic to the Neotropics, mostly distributed in the Atlantic coastal forests of Brazil. Here the first comprehensive phylogenetic study of this group is presented, and this phylogeny is used as the basis to evaluate the recent infrageneric classification in Eugenia sensu lato (s.l.) to test the history of the evolution of traits in the group and test hypotheses associated with the history of this clade. METHODS: A total of 42 taxa were sampled, of which 14 were Eugenia sect. Phyllocalyx for one nuclear (ribosomal internal transcribed spacer) and four plastid markers (psbA-trnH, rpl16, trnL-rpl32 and trnQ-rps16). The relationships were reconstructed based on Bayesian analysis and maximum likelihood. Additionally, ancestral area analysis and modelling methods were used to estimate species dispersal, comparing historically climatic stable (refuges) and unstable areas. KEY RESULTS: Maximum likelihood and Bayesian inferences indicate that Eugenia sect. Phyllocalyx is paraphyletic and the two clades recovered are characterized by combinations of morphological characters. Phylogenetic relationships support a link between Cerrado and south-eastern species and a difference in the composition of species from north-eastern and south-eastern Atlantic forest. Refugia and stable areas identified within unstable areas suggest that these areas were important to maintain diversity in the Atlantic forest biodiversity hotspot. CONCLUSION: This study provides a robust phylogenetic framework to address important historical questions for Eugenia s.l. within an evolutionary context, supporting the need for better taxonomic study of one of the largest genera in the Neotropics. Furthermore, valuable insight is offered into diversification and biome shifts of plant species in the highly environmentally impacted Atlantic forest of South America. Evidence is presented that climate stability in the south-eastern Atlantic forest during the Quaternary contributed to the highest levels of plant diversity in this region that acted as a refugium.

Environmental variations drive polyploid evolution in neotropical Eugenia species (Myrtaceae).

Polyploidy is one of the most important mechanisms of speciation and diversification in plant evolution. Polyploidy results in genetic variation among individuals of the same species and even between populations, and may be responsible for differences in environmental tolerance between populations of the same species. This study determined chromosome numbers of Eugenia L. (Myrtaceae, x = 11) for 26 populations of 14 species by conventional cytogenetic techniques. Nine species (13 populations) were diploid (2n = 2x = 22), but diploid and/or polyploid cytotypes were found in the other five species (13 populations), with 2n = 33, 2n = 44, and 2n = 55. Data on chromosome number/ploidy level for other Eugenia species/populations were collected from the literature and included in this cytogeographic analysis. For each collection point (32 species and 62 populations), environmental variables were recorded using georeferencing techniques through the DIVA-GIS v.7.5 program. Environmental variables such as temperature, altitude, rainfall, solar radiation, soil type, and vegetation were analyzed with the R program, using Mann-Whitney and chi-square tests, principal component analysis, and graphic analyses, such as scatterplots, boxplots, and barplot. Polyploid and diploid populations had different spatial distribution patterns and were found in areas subjected to different environmental conditions. Polyploid individuals were collected from locations with more adverse environmental conditions, usually at higher elevations than the diploid individuals. Polyploidy allows species to occur at locations with varying environmental conditions. As diploidy and polyploidy occur under different environmental conditions, species with cytotypes exhibit wide environmental tolerance.

Mating system and pollen dispersal in Eugenia dysenterica (Myrtaceae) germplasm collection: tools for conservation and domestication.

Eugenia dysenterica DC. (Myrtaceae) is a perennial tree producing edible fruits and ornamental flowers of potential value widely distributed in Brazilian "Cerrados" (savannas), but available genetic resources and potential for future breeding programs must be evaluated. Here we evaluated the reproductive system and pollen-mediated gene flow in one generation of Eugenia dysenterica germplasm collection of Agronomy School, Federal University of Goiás (in Goiânia city, Central Brazil). We collected leaves from all adults from the germplasm collection (682 plants) and seeds (542) from 23 mother-trees. Genotypes were obtained for seven microsatellite loci. Genetic diversity was high and did not significantly differ between adults (H e = 0.777) and progeny arrays (H e = 0.617). Our results showed that E. dysenterica has an allogamous mating system in the germplasm collection (t m = 0.957), but with high and significant biparental inbreeding (t m - t s = 0.109). Because sibs are very close to each other, mating between closely related individuals is likely. Paternity correlation was also relatively high, indicating a 11.9 % probability that a randomly chosen pair of outcrossed progeny from the same array are full sibs. The maximum pollen dispersal distance (224 m), estimated using assignment test, corresponded to the boundaries of the orchard. We were able to assign the paternity to only 64 % of the 349 seeds analyzed, indicating potential pollen immigration to the germplasm collection. The variance effective population size estimated for one maternal family in the germplasm collection (N ev = 3.42) is very close to the theoretical maximum value for half-sibs (Nev = 4.0). Because E. dysenterica has a long life cycle and generation time, the maintenance of an effective population size of at least 100 in the germplasm collection is suggested, which can be achieved by maintaining a seed-trees number around 30 individuals.

Exhaustive search for conservation networks of populations representing genetic diversity.

Conservation strategies routinely use optimization methods to identify the smallest number of units required to represent a set of features that need to be conserved, including biomes, species, and populations. In this study, we provide R scripts to facilitate exhaustive search for solutions that represent all of the alleles in networks with the smallest possible number of populations. The script also allows other variables to be added to describe the populations, thereby providing the basis for multi-objective optimization and the construction of Pareto curves by averaging the values in the solutions. We applied this algorithm to an empirical dataset that comprised 23 populations of Eugenia dysenterica, which is a tree species with a widespread distribution in the Cerrado biome. We observed that 15 populations would be necessary to represent all 249 alleles based on 11 microsatellite loci, and that the likelihood of representing all of the alleles with random networks is less than 0.0001. We selected the solution (from two with the smallest number of populations) obtained for the populations with a higher level of climatic stability as the best strategy for in situ conservation of genetic diversity of E. dysenterica. The scripts provided in this study are a simple and efficient alternative to more complex optimization methods, especially when the number of populations is relatively small (i.e., <25 populations).

Evaluation of antifungal activity in essential oil of the Syzygium aromaticum (L) by extraction, purification and analysis of its main component eugenol

Antifungal properties of some essential oils have been well documented. Clove oil is reported to have strong antifungal activity against many fungal species. In this study we have evaluated antifungal potential of essential oil of Syzygium aromaticum (L.) against some common fungal pathogens of plants and animals namely, Fusarium moniliforme NCIM 1100, Fusarium oxysporum MTCC 284, Aspergillus sp., Mucor sp., Trichophyton rubrum and Microsporum gypseum. All fungal species were found to be inhibited by the oil when tested through agar well diffusion method. Minimum inhibitory concentration (MIC) was determined for all the species. Column chromatography was performed to separate the eugenol rich fraction from clove oil. Out of seven fractions maximum activity was obtained in column fraction II. TLC and HPLC data confirmed presence of considerable Eugenol in fraction II and clove oil. Microscopic study on effect of clove oil and column fraction II on spores of Mucor sp. and M. gypseum showed distortion and shrinkage while it was absent in other column fractions. So it can be concluded that the antifungal action of clove oil is due to its high eugenol content.

Genetic structure of natural Eugenia dysenterica DC (Myrtaceae) populations in northeastern Goiás, Brazil, accessed by morphological traits and RAPD markers

The Brazilian Cerrado (Savannah) is a biome with great biodiversity. Many of the species found in this biome have unlimited economic potential and are used by the local population for various purposes. With the opening of the agricultural frontiers in this region, much of the native flora has been replaced by extensive areas of monocultures. Thirteen populations of the Eugenia dysenterica DC species were sampled in the northeast of the state of Goiás, Brazil, to analyze their genetic structure. Morphological traits were measured for all the progeny obtained and some of them were analyzed by using RAPD markers. The natural populations of the species showed high levels of variability for the morphological traits. The phenotypic, genotypic and genetic variabilities as structured in these populations were highly significant among populations, although most of the variability was detected within populations. The phenotypic and genetic variabilities were structured spatially as shown by the positive and highly significant correlation coefficients between the phenotypic and genetic distance matrixes and the geographic distance matrix. The ideal conservation strategy for the species in the region requires sampling of a high number of individuals of each population and also a significant number of populations to ensure suitable effective population sizes.