Felipe Rodrigues da Silva

InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams

BackgroundSet comparisons permeate a large number of data analysis workflows, in particular workflows in biological sciences. Venn diagrams are frequently employed for such analysis but current tools are limited.ResultsWe have developed InteractiVenn, a more flexible tool for interacting with Venn diagrams including up to six sets. It offers a clean interface for Venn diagram construction and enables analysis of set unions while preserving the shape of the diagram. Set unions are useful to reveal differences and similarities among sets and may be guided in our tool by a tree or by a list of set unions. The tool also allows obtaining subsets’ elements, saving and loading sets for further analyses, and exporting the diagram in vector and image formats. InteractiVenn has been used to analyze two biological datasets, but it may serve set analysis in a broad range of domains.ConclusionsInteractiVenn allows set unions in Venn diagrams to be explored thoroughly, by consequence extending the ability to analyze combinations of sets with additional observations, yielded by novel interactions between joined sets. InteractiVenn is freely available online at: www.interactivenn.net.

Brazilian coffee genome project: an EST-based genomic resource

Coffee is one of the most valuable agricultural commodities and ranks second on international trade exchanges. The genus Coffea belongs to the Rubiaceae family which includes other important plants. The genus contains about 100 species but commercial production is based only on two species, Coffea arabica and Coffea canephora that represent about 70 % and 30 % of the total coffee market, respectively. The Brazilian Coffee Genome Project was designed with the objective of making modern genomics resources available to the coffee scientific community, working on different aspects of the coffee production chain. We have single-pass sequenced a total of 214,964 randomly picked clones from 37 cDNA libraries of C. arabica, C. canephora and C. racemosa, representing specific stages of cells and plant development that after trimming resulted in 130,792, 12,381 and 10,566 sequences for each species, respectively. The ESTs clustered into 17,982 clusters and 32,155 singletons. Blast analysis of these sequences revealed that 22 % had no significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function). The generated coffee EST database resulted in the identification of close to 33,000 different unigenes. Annotated sequencing results have been stored in an online database at http://www.lge.ibi.unicamp.br/cafe. Resources developed in this project provide genetic and genomic tools that may hold the key to the sustainability, competitiveness and future viability of the coffee industry in local and international markets.

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

BackgroundBrachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.ResultsEight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were BbrizUBCE, BbrizE1F4A and BbrizEF1.ConclusionIn total, eight genes belonging to different cellular processes were tested. Out of them, BbrizTUB was the less stable while BbrizEF1 followed by BbrizUBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were BbrizUBCE, BbrizE1F4A and BbrizEF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.

ESTs from a wild Arachis species for gene discovery and marker development

BackgroundDue to its origin, peanut has a very narrow genetic background. Wild relatives can be a source of genetic variability for cultivated peanut. In this study, the transcriptome of the wild species Arachis stenosperma accession V10309 was analyzed.ResultsESTs were produced from four cDNA libraries of RNAs extracted from leaves and roots of A. stenosperma. Randomly selected cDNA clones were sequenced to generate 8,785 ESTs, of which 6,264 (71.3%) had high quality, with 3,500 clusters: 963 contigs and 2537 singlets. Only 55.9% matched homologous sequences of known genes. ESTs were classified into 23 different categories according to putative protein functions. Numerous sequences related to disease resistance, drought tolerance and human health were identified. Two hundred and six microsatellites were found and markers have been developed for 188 of these. The microsatellite profile was analyzed and compared to other transcribed and genomic sequence data.ConclusionThis is, to date, the first report on the analysis of transcriptome of a wild relative of peanut. The ESTs produced in this study are a valuable resource for gene discovery, the characterization of new wild alleles, and for marker development. The ESTs were released in the [GenBank:EH041934 to EH048197].

Identification of drought-responsive genes in roots of upland rice (Oryza sativa L)

BackgroundRice (Oryza sativa L.) germplasm represents an extraordinary source of genes that control traits of agronomic importance such as drought tolerance. This diversity is the basis for the development of new cultivars better adapted to water restriction conditions, in particular for upland rice, which is grown under rainfall. The analyses of subtractive cDNA libraries and differential protein expression of drought tolerant and susceptible genotypes can contribute to the understanding of the genetic control of water use efficiency in rice.ResultsTwo subtractive libraries were constructed using cDNA of drought susceptible and tolerant genotypes submitted to stress against cDNA of well-watered plants. In silico analysis revealed 463 reads, which were grouped into 282 clusters. Several genes expressed exclusively in the tolerant or susceptible genotypes were identified. Additionally, proteome analysis of roots from stressed plants was performed and 22 proteins putatively associated to drought tolerance were identified by mass spectrometry.ConclusionSeveral genes and proteins involved in drought-response, as well as genes with no described homologs were identified. Genes exclusively expressed in the tolerant genotype were, in general, related to maintenance of turgor and cell integrity. In contrast, in the susceptible genotype, expression of genes involved in protection against cell damage was not detected. Several protein families identified in the proteomic analysis were not detected in the cDNA analysis. There is an indication that the mechanisms of susceptibility to drought in upland rice are similar to those of lowland varieties.

Insights into the Musa genome: Syntenic relationships to rice and between Musa species

BackgroundMusa species (Zingiberaceae, Zingiberales) including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome.ResultsWe produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales) orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya.ConclusionThese results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic traits for breeding purposes.

Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans

Several Brazilian sugarcane varieties have the ability to grow with little addition of inorganic nitrogen fertilizers, showing high contributions of Biological Nitrogen Fixation (BNF). A particular type of nitrogen-fixing association has been described in this crop, where endophytic diazotrophs such as Gluconacetobacter diazotrophicus and Herbaspirillum spp. colonize plant tissues without causing disease symptoms. In order to gain insight into the role played by the sugarcane in the interaction between this plant and endophytic diazotrophs, we investigated gene expression profiles of sugarcane plants colonized by G. diazotrophicus and H. rubrisubalbicans by searching the sugarcane expressed sequence tag SUCEST Database (http://sucest.lad.ic.unicamp.br/en/). We produced an inventory of sugarcane genes, candidates for exclusive or preferential expression during the nitrogen-fixing association. This data suggests that the host plant might be actively involved in the establishment of the interaction with G. diazotrophicus and H. rubrisubalbicans.

Trimming and clustering sugarcane ESTs

The original clustering procedure adopted in the Sugarcane Expressed Sequence Tag project (SUCEST) had many problems, for instance too many clusters, the presence of ribosomal sequences, etc. We therefore redesigned the clustering procedure entirely, including a much more careful initial trimming of the reads. In this paper the new trimming and clustering strategies are described in detail and we give the new official figures for the project, 237,954 expressed sequence tags and 43,141 clusters.

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Transcriptional profile of maize roots under acid soil growth

BackgroundAluminum (Al) toxicity is one of the most important yield-limiting factors of many crops worldwide. The primary symptom of Al toxicity syndrome is the inhibition of root growth leading to poor water and nutrient absorption. Al tolerance has been extensively studied using hydroponic experiments. However, unlike soil conditions, this method does not address all of the components that are necessary for proper root growth and development. In the present study, we grew two maize genotypes with contrasting tolerance to Al in soil containing toxic levels of Al and then compared their transcriptomic responses.ResultsWhen grown in acid soil containing toxic levels of Al, the Al-sensitive genotype (S1587-17) showed greater root growth inhibition, more Al accumulation and more callose deposition in root tips than did the tolerant genotype (Cat100-6). Transcriptome profiling showed a higher number of genes differentially expressed in S1587-17 grown in acid soil, probably due to secondary effects of Al toxicity. Genes involved in the biosynthesis of organic acids, which are frequently associated with an Al tolerance response, were not differentially regulated in both genotypes after acid soil exposure. However, genes related to the biosynthesis of auxin, ethylene and lignin were up-regulated in the Al-sensitive genotype, indicating that these pathways might be associated with root growth inhibition. By comparing the two maize lines, we were able to discover genes up-regulated only in the Al-tolerant line that also presented higher absolute levels than those observed in the Al-sensitive line. These genes encoded a lipase hydrolase, a retinol dehydrogenase, a glycine-rich protein, a member of the WRKY transcriptional family and two unknown proteins.ConclusionsThis work provides the first characterization of the physiological and transcriptional responses of maize roots when grown in acid soil containing toxic levels of Al. The transcriptome profiles highlighted several pathways that are related to Al toxicity and tolerance during growth in acid soil. We found several genes that were not found in previous studies using hydroponic experiments, increasing our understanding of plant responses to acid soil. The use of two germplasms with markedly different Al tolerances allowed the identification of genes that are a valuable tool for assessing the mechanisms of Al tolerance in maize in acid soil.