The correct identification of differentially expressed genes (DEGs) between specific conditions is a key in the understanding phenotypic variation. High-throughput transcriptome sequencing (RNA-Seq) has become the main option for these studies. Thus, the number of methods and softwares for differential expression analysis from RNA-Seq data also increased rapidly. However, there is no consensus about the most appropriate pipeline or protocol for identifying differentially expressed genes from RNA-Seq data. This work presents an extended review on the topic that includes the evaluation of six methods of mapping reads, including pseudo-alignment and quasi-mapping and nine methods of differential expression analysis from RNA-Seq data. The adopted methods were evaluated based on real RNA-Seq data, using qRT-PCR data as reference (gold-standard). As part of the results, we developed a software that performs all the analysis presented in this work, which is freely available at https://github.com/costasilvati/consexpression. The results indicated that mapping methods have minimal impact on the final DEGs analysis, considering that adopted data have an annotated reference genome. Regarding the adopted experimental model, the DEGs identification methods that have more consistent results were the limma+voom, NOIseq and DESeq2. Additionally, the consensus among five DEGs identification methods guarantees a list of DEGs with great accuracy, indicating that the combination of different methods can produce more suitable results. The consensus option is also included for use in the available software.

BackgroundSugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out.ResultsSixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements.ConclusionsIndividual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

Plant exposure to abiotic stresses leads to an accumulation of reactive oxygen species with the concomitant increase in antioxidant defense mechanisms. Previous studies showed that exogenous application of proline mitigate the deleterious effects caused by oxidative stress due to its ability to increase the activity of antioxidant enzymes. However, there are no reports of the effects of high endogenous accumulation of proline in the transcriptional pattern of antioxidant enzymes genes under normal conditions of water supply or in response to water deficit. Here, we show that isoforms of four antioxidant enzymes genes (Ascorbate peroxidase-APX, Catalase-CAT, Superoxide dismutase-SOD and Glutathione reductase-GR) were differentially regulated in leaves of Swingle citrumelo transgenic plants with high endogenous proline accumulation submitted to water deficits and also under normal water supply condition. Proline per se caused a two-fold change in the transcription activity of APX1, APXcl, CAT2 and Cu/ZnSOD2, while during water deficit proline influenced mRNAs levels in APXs and Cu/ZnSODs isoforms, MnSODmit and GRcl. This study adds new information on the role of proline during drought conditions and, more important, without the potential confounding effects imposed by water deficiency. We showed that, in addition to its known effects on diverse plant physiological and biochemical processes, high endogenous proline can also acts as a regulatory/signalling molecule capable of altering the transcript levels of stress-related genes.

Genotypic, developmental, and environmental factors converge to determine the degree of Crassulacean acid metabolism (CAM) expression. To characterize the signaling events controlling CAM expression in young pineapple (Ananas comosus) plants, this photosynthetic pathway was modulated through manipulations in water availability. Rapid, intense, and completely reversible up-regulation in CAM expression was triggered by water deficit, as indicated by the rise in nocturnal malate accumulation and in the expression and activity of important CAM enzymes. During both up-and down-regulation of CAM, the degree of CAM expression was positively and negatively correlated with the endogenous levels of abscisic acid (ABA) and cytokinins, respectively. When exogenously applied, ABA stimulated and cytokinins repressed the expression of CAM. However, inhibition of water deficit-induced ABA accumulation did not block the up-regulation of CAM, suggesting that a parallel, non-ABA-dependent signaling route was also operating. Moreover, strong evidence revealed that nitric oxide (NO) may fulfill an important role during CAM signaling. Up-regulation of CAM was clearly observed in NO-treated plants, and a conspicuous temporal and spatial correlation was also evident between NO production and CAM expression. Removal of NO from the tissues either by adding NO scavenger or by inhibiting NO production significantly impaired ABA-induced up-regulation of CAM, indicating that NO likely acts as a key downstream component in the ABA-dependent signaling pathway. Finally, tungstate or glutamine inhibition of the NO-generating enzyme nitrate reductase completely blocked NO production during ABA-induced up-regulation of CAM, characterizing this enzyme as responsible for NO synthesis during CAM signaling in pineapple plants.

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main São Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in São Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

Lipids, including the diterpenes cafestol and kahweol, are key compounds that contribute to the quality of coffee beverages. We determined total lipid content and cafestol and kahweol concentrations in green beans and genotyped 107 Coffea arabica accessions, including wild genotypes from the historical FAO collection from Ethiopia. A genome-wide association study was performed to identify genomic regions associated with lipid, cafestol and kahweol contents and cafestol/kahweol ratio. Using the diploid Coffea canephora genome as a reference, we identified 6,696 SNPs. Population structure analyses suggested the presence of two to three groups (K = 2 and K = 3) corresponding to the east and west sides of the Great Rift Valley and an additional group formed by wild accessions collected in western forests. We identified 5 SNPs associated with lipid content, 4 with cafestol, 3 with kahweol and 9 with cafestol/kahweol ratio. Most of these SNPs are located inside or near candidate genes related to metabolic pathways of these chemical compounds in coffee beans. In addition, three trait-associated SNPs showed evidence of directional selection among cultivated and wild coffee accessions. Our results also confirm a great allelic richness in wild accessions from Ethiopia, especially in accessions originating from forests in the west side of the Great Rift Valley.

On the world stage, the increase in temperatures due to global warming is already a reality that has become one of the main challenges faced by the scientific community. Since agriculture is highly dependent on climatic conditions, it may suffer a great impact in the short term if no measures are taken to adapt and mitigate the agricultural system. Plant responses to abiotic stresses have been the subject of research by numerous groups worldwide. Initially, these studies were concentrated on model plants, and, later, they expanded their studies in several economically important crops such as rice, corn, soybeans, coffee, and others. However, agronomic evaluations for the launching of cultivars and the classical genetic improvement process focus, above all, on productivity, historically leaving factors such as tolerance to abiotic stresses in the background. Considering the importance of the impact that abiotic stresses can have on agriculture in the short term, new strategies are currently being sought and adopted in breeding programs to understand the physiological, biochemical, and molecular responses to environmental disturbances in plants of agronomic interest, thus ensuring the world food security. Moreover, integration of these approaches is bringing new insights on breeding. We will discuss how water deficit, high temperatures, and salinity exert effects on plants.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.