BrumiR: A toolkit for de novo discovery of microRNAs from sRNA-seq data.

MicroRNAs (miRNAs) are small noncoding RNAs that are key players in the regulation of gene expression. In the past decade, with the increasing accessibility of high-throughput sequencing technologies, different methods have been developed to identify miRNAs, most of which rely on preexisting reference genomes. However, when a reference genome is absent or is not of high quality, such identification becomes more difficult. In this context, we developed BrumiR, an algorithm that is able to discover miRNAs directly and exclusively from small RNA (sRNA) sequencing (sRNA-seq) data. We benchmarked BrumiR with datasets encompassing animal and plant species using real and simulated sRNA-seq experiments. The results demonstrate that BrumiR reaches the highest recall for miRNA discovery, while at the same time being much faster and more efficient than the state-of-the-art tools evaluated. The latter allows BrumiR to analyze a large number of sRNA-seq experiments, from plants or animal species. Moreover, BrumiR detects additional information regarding other expressed sequences (sRNAs, isomiRs, etc.), thus maximizing the biological insight gained from sRNA-seq experiments. Additionally, when a reference genome is available, BrumiR provides a new mapping tool (BrumiR2reference) that performs an a posteriori exhaustive search to identify the precursor sequences. Finally, we also provide a machine learning classifier based on a random forest model that evaluates the sequence-derived features to further refine the prediction obtained from the BrumiR-core. The code of BrumiR and all the algorithms that compose the BrumiR toolkit are freely available at https://github.com/camoragaq/BrumiR.

Plant ecological genomics at the limits of life in the Atacama Desert.

The Atacama Desert in Chile-hyperarid and with high-ultraviolet irradiance levels-is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre-Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil-microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. We deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a "genetic goldmine" to engineer crop resilience to face climate change.

Shotgun proteomics of peach fruit reveals major metabolic pathways associated to ripening.

BACKGROUND: Fruit ripening in Prunus persica melting varieties involves several physiological changes that have a direct impact on the fruit organoleptic quality and storage potential. By studying the proteomic differences between the mesocarp of mature and ripe fruit, it would be possible to highlight critical molecular processes involved in the fruit ripening. RESULTS: To accomplish this goal, the proteome from mature and ripe fruit was assessed from the variety O'Henry through shotgun proteomics using 1D-gel (PAGE-SDS) as fractionation method followed by LC/MS-MS analysis. Data from the 131,435 spectra could be matched to 2740 proteins, using the peach genome reference v1. After data pre-treatment, 1663 proteins could be used for comparison with datasets assessed using transcriptomic approaches and for quantitative protein accumulation analysis. Close to 26% of the genes that code for the proteins assessed displayed higher expression at ripe fruit compared to other fruit developmental stages, based on published transcriptomic data. Differential accumulation analysis between mature and ripe fruit revealed that 15% of the proteins identified were modulated by the ripening process, with glycogen and isocitrate metabolism, and protein localization overrepresented in mature fruit, as well as cell wall modification in ripe fruit. Potential biomarkers for the ripening process, due to their differential accumulation and gene expression pattern, included a pectin methylesterase inhibitor, a gibbellerin 2-beta-dioxygenase, an omega-6 fatty acid desaturase, a homeobox-leucine zipper protein and an ACC oxidase. Transcription factors enriched in NAC and Myb protein domains would target preferentially the genes encoding proteins more abundant in mature and ripe fruit, respectively. CONCLUSIONS: Shotgun proteomics is an unbiased approach to get deeper into the proteome allowing to detect differences in protein abundance between samples. This technique provided a resolution so that individual gene products could be identified. Many proteins likely involved in cell wall and sugar metabolism, aroma and color, change their abundance during the transition from mature to ripe fruit.

Mycoplasma hyopneumoniae J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells.

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

Whole Genome Sequence, Variant Discovery and Annotation in Mapuche-Huilliche Native South Americans.

Whole human genome sequencing initiatives help us understand population history and the basis of genetic diseases. Current data mostly focuses on Old World populations, and the information of the genomic structure of Native Americans, especially those from the Southern Cone is scant. Here we present annotation and variant discovery from high-quality complete genome sequences of a cohort of 11 Mapuche-Huilliche individuals (HUI) from Southern Chile. We found approximately 3.1 × 106 single nucleotide variants (SNVs) per individual and identified 403,383 (6.9%) of novel SNVs events. Analyses of large-scale genomic events detected 680 copy number variants (CNVs) and 4,514 structural variants (SVs), including 398 and 1,910 novel events, respectively. Global ancestry composition of HUI genomes revealed that the cohort represents a sample from a marginally admixed population from the Southern Cone, whose main genetic component derives from Native American ancestors. Additionally, we found that HUI genomes contain variants in genes associated with 5 of the 6 leading causes of noncommunicable diseases in Chile, which may have an impact on the risk of prevalent diseases in Chilean and Amerindian populations. Our data represents a useful resource that can contribute to population-based studies and for the design of early diagnostics or prevention tools for Native and admixed Latin American populations.

Overview of Nucleotide Sugar Transporter Gene Family Functions Across Multiple Species.

Glycoproteins and glycolipids are crucial in a number of cellular processes, such as growth, development, and responses to external cues, among others. Polysaccharides, another class of sugar-containing molecules, also play important structural and signaling roles in the extracellular matrix. The additions of glycans to proteins and lipids, as well as polysaccharide synthesis, are processes that primarily occur in the Golgi apparatus, and the substrates used in this biosynthetic process are nucleotide sugars. These proteins, lipids, and polysaccharides are also modified by the addition of sulfate groups in the Golgi apparatus in a series of reactions where nucleotide sulfate is needed. The required nucleotide sugar substrates are mainly synthesized in the cytosol and transported into the Golgi apparatus by nucleotide sugar transporters (NSTs), which can additionally transport nucleotide sulfate. Due to the critical role of NSTs in eukaryotic organisms, any malfunction of these could change glycan and polysaccharide structures, thus affecting function and altering organism physiology. For example, mutations or deletion on NST genes lead to pathological conditions in humans or alter cell walls in plants. In recent years, many NSTs have been identified and functionally characterized, but several remain unanalyzed. This study examined existing information on functionally characterized NSTs and conducted a phylogenetic analysis of 257 NSTs predicted from nine animal and plant model species, as well as from protists and fungi. From this analysis, relationships between substrate specificity and the primary NST structure can be inferred, thereby advancing understandings of nucleotide sugar gene family functions across multiple species.

Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait.

Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients.

Whole genome comparison between table and wine grapes reveals a comprehensive catalog of structural variants.

BACKGROUND: Grapevine (Vitis vinifera L.) is the most important Mediterranean fruit crop, used to produce both wine and spirits as well as table grape and raisins. Wine and table grape cultivars represent two divergent germplasm pools with different origins and domestication history, as well as differential characteristics for berry size, cluster architecture and berry chemical profile, among others. 'Sultanina' plays a pivotal role in modern table grape breeding providing the main source of seedlessness. This cultivar is also one of the most planted for fresh consumption and raisins production. Given its importance, we sequenced it and implemented a novel strategy for the de novo assembly of its highly heterozygous genome. RESULTS: Our approach produced a draft genome of 466 Mb, recovering 82% of the genes present in the grapevine reference genome; in addition, we identified 240 novel genes. A large number of structural variants and SNPs were identified. Among them, 45 (21 SNPs and 24 INDELs) were experimentally confirmed in 'Sultanina' and six SNPs in other 23 table grape varieties. Transposable elements corresponded to ca. 80% of the repetitive sequences involved in structural variants and more than 2,000 genes were affected in their structure by these variants. Some of these genes are likely involved in embryo development, suggesting that they may contribute to seedlessness, a key trait for table grapes. CONCLUSIONS: This work produced the first structural variants and SNPs catalog for grapevine, constituting a novel and very powerful tool for genomic studies in this key fruit crop, particularly useful to support marker assisted breeding in table grapes.