Mauricio Pereira Almerão

Identification of novel soybean microRNAs involved in abiotic and biotic stresses

BackgroundSmall RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.ResultsThe libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.ConclusionsValidation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.

Identifying conserved and novel microRNAs in developing seeds of Brassica napus using deep sequencing.

MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. Members of 59 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally 29 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. Assembled mRNA-seq contigs allowed for a search of putative new precursors and led to the identification of 13 novel miRNA families. Analysis of miRNA population between libraries reveals that several miRNAs and isomiRNAs have different abundance in developing stages compared to mature seeds. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comparative study of the miRNA transcriptome of mature and developing B. napus seeds and provides a basis for future research on individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus.

Identification of microRNAs from Eugenia uniflora by high-throughput sequencing and bioinformatics analysis.

Background microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. Results Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. Conclusions This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

Terrestrial isopod diversity in the subtropical Neotropics: Itapuã State Park, southern Brazil

We analysed species abundance and composition during one year of sampling at Itapua State Park, a conservation unit in the State of Rio Grande do Sul, southern Brazil. Samples were collected monthly from May 2003 to April 2004 along a 4 km forest trail divided in three transects along the slopes of a small hill. Terrestrial isopods were hand searched by three people for 40 minutes at two sampling sites along each transect, summing 240 minutes of sampling effort per transect per month. Six species distributed in six families were found. The collector curve stabilised when half the samples were taken, demonstrating sampling sufficiency: analytical estimates did not predict more species to be found in the trail. Atlantoscia floridana (van Name, 1940) was dominant in all transects. Abundance varied along the year with a summer and a winter peak, but peaks were not consistent among transects. The first part of the trail, used for ecotourism, was less diverse, had less species (4 as compared to 6 in the other transects), and was thus also less similar in composition.

Checklist of the Crustacea from the state of Santa Catarina, Brazil

The list of Crustacean species from the state of Santa Catarina, southern Brazil, comprises 518 valid species, distributed in 17 orders and 152 families. This amount corresponds to 0.75% of the Crustacea worldwide and about 20% of Brazilian species. The order Decapoda is the most represented, with 280 species. The families of Decapoda with the greatest number of species recorded were: Aeglidae (19), Palaemonidae (13) and Portunidae (13). In addition, the occurrence of 19 species distributed in the orders Cyclopoida (1), Podocopida (2) and Decapoda (16) is reported for the first time. Exotic species (9) are distributed in four different orders - Isopoda (6), Calanoida (1), Thoracica (1) and Decapoda (1).

Red swamp crayfish: biology, ecology and invasion - an overview

Alien species have been transported and traded by humans for many centuries. However, with the era of globalization, biological invasions have reached notable magnitudes. Currently, introduction of alien species is one of the major threats to biodiversity and ecosystem functioning. The North American crayfish Procambarus clarkii is one of the most widely introduced freshwater species in the world, especially due to its high economic importance. It is responsible for great modifications in invaded environments causing irreparable ecological and economic damages. Its impressive ability to successfully colonize a wide range of environments is a consequence of its behavioural and biological characteristics that can adapt to features of the invaded location, conferring to this species a notable ecological plasticity. This review summarizes the available information regarding P. clarkiis biology and invasive dynamics around the world in order to contribute to the understanding of the threats posed by its establishment, as well as to support management and impact mitigation efforts.

Distribution, introduction pathway, and invasion risk analysis of the North American crayfish Procambarus clarkii (Decapoda: Cambaridae) in southeast Brazil.

This study reports new records of Procambarus clarkii Girard, 1852 in Southeastern Brazil and investigates its possible pathways of introduction. The Brazilian crayfish pet trade was surveyed with regard to species’ availability and price, reporting its persistence, even though illegal. An invasion risk analysis was performed by applying a protocol developed specifically to invasive freshwater invertebrates (FI-ISK). Procambarus clarkii has confirmed occurrences from urban and non-urban areas in the state of Sao Paulo, including the Jaragua State Park, an Atlantic Forest conservation area. The pet trade is an important introduction vector but some subsequent translocation can also be due to the release of live bait by anglers. Besides passive dispersal, these newly discovered populations, especially those located in non-urban areas, are likely to naturally disperse further through active spread and river drift of juveniles. Although confirmed colonized areas are all located in the state of Sao Paulo, the invasive risk analysis showed that P. clarkii is a potential highly invasive species for Brazil. These introductions have unknown consequences; however, many impacts are associated with this species establishment around the world, underlining the urgency to understand the invasion process in Brazil and develop efficient management techniques.

Identification of potential miRNAs and their targets in Vriesea carinata (Poales, Bromeliaceae)

The miRNAs play important roles in regulation of gene expression at the post-transcriptional level. A small RNA and RNA-seq of libraries were constructed to identify miRNAs in Vriesea carinata, a native bromeliad species from Brazilian Atlantic Rainforest. Illumina technology was used to perform high throughput sequencing and data was analyzed using bioinformatics tools. We obtained 2,191,509 mature miRNAs sequences representing 54 conserved families in plant species. Further analysis allowed the prediction of secondary structures for 19 conserved and 16 novel miRNAs. Potential targets were predicted from pre-miRNAs by sequence homology and validated using RTqPCR approach. This study provides the first identification of miRNAs and their potential targets of a bromeliad species.

First record of Wolbachia in South American terrestrial isopods: Prevalence and diversity in two species of Balloniscus (Crustacea, Oniscidea)

Wolbachia are endosymbiotic bacteria that commonly infect arthropods, inducing certain phenotypes in their hosts. So far, no endemic South American species of terrestrial isopods have been investigated for Wolbachia infection. In this work, populations from two species of Balloniscus (B. sellowii and B. glaber) were studied through a diagnostic PCR assay. Fifteen new Wolbachia 16S rDNA sequences were detected. Wolbachia found in both species were generally specific to one population, and five populations hosted two different Wolbachia 16S rDNA sequences. Prevalence was higher in B. glaber than in B. sellowii, but uninfected populations could be found in both species. Wolbachia strains from B. sellowii had a higher genetic variation than those isolated from B. glaber. AMOVA analyses showed that most of the genetic variance was distributed among populations of each species rather than between species, and the phylogenetic analysis suggested that Wolbachia strains from Balloniscus cluster within Supergroup B, but do not form a single monophyletic clade, suggesting multiple infections for this group. Our results highlight the importance of studying Wolbachia prevalence and genetic diversity in Neotropical species and suggest that South American arthropods may harbor a great number of diverse strains, providing an interesting model to investigate the evolution of Wolbachia and its hosts.

Nova espécie de Trichorhina (Isopoda, Oniscidea, Plathyarthridae) do Brasil

A new species of Trichorhina (Isopoda, Oniscidea, Plathyarthridae) from Brazil. Trichorhina tatianae sp. nov., found during recent surveys in Ararangua, Morro dos Conventos, in the State of Santa Catarina, is described herein. The new species can easily be differentiated by the lateral expansion of the ischium of pereiopod 7 of the male. Trichorhina tatianae sp. nov. is recorded only from its type-locality.