The Soybean Expression Atlas v2: A comprehensive database of over 5000 RNA-seq samples.

Soybean is a crucial crop worldwide, used as a source of food, feed, and industrial products due to its high protein and oil content. Previously, the rapid accumulation of soybean RNA-seq data in public databases and the computational challenges of processing raw RNA-seq data motivated us to develop the Soybean Expression Atlas, a gene expression database of over a thousand RNA-seq samples. Over the past few years, our database has allowed researchers to explore the expression profiles of important gene families, discover genes associated with agronomic traits, and understand the transcriptional dynamics of cellular processes. Here, we present the Soybean Expression Atlas v2, an updated version of our database with a fourfold increase in the number of samples, featuring transcript- and gene-level transcript abundance matrices for 5481 publicly available RNA-seq samples. New features in our database include the availability of transcript-level abundance estimates and equivalence classes to explore differential transcript usage, abundance estimates in bias-corrected counts to increase the accuracy of differential gene expression analyses, a new web interface with improved data visualization and user experience, and a reproducible and scalable pipeline available as an R package. The Soybean Expression Atlas v2 is available at https://soyatlas.venanciogroup.uenf.br/, and it will accelerate soybean research, empowering researchers with high-quality and easily accessible gene expression data.

Comparative Genomics Reveals Novel Species and Insights into the Biotechnological Potential, Virulence, and Resistance of Alcaligenes.

Alcaligenes is a cosmopolitan bacterial genus that exhibits diverse properties which are beneficial to plants. However, the genomic versatility of Alcaligenes has also been associated with the ability to cause opportunistic infections in humans, raising concerns about the safety of these microorganisms in biotechnological applications. Here, we report an in-depth comparative analysis of Alcaligenes species using all publicly available genomes to investigate genes associated with species, biotechnological potential, virulence, and resistance to multiple antibiotics. Phylogenomic analysis revealed that Alcaligenes consists of at least seven species, including three novel species. Pan-GWAS analysis uncovered 389 species-associated genes, including cold shock proteins (e.g., cspA) and aquaporins (e.g., aqpZ) found exclusively in the water-isolated species, Alcaligenes aquatilis. Functional annotation of plant-growth-promoting traits revealed enrichment of genes for auxin biosynthesis, siderophores, and organic acids. Genes involved in xenobiotic degradation and toxic metal tolerance were also identified. Virulome and resistome profiles provide insights into selective pressures exerted in clinical settings. Taken together, the results presented here provide the grounds for more detailed clinical and ecological studies of the genus Alcaligenes.

Molecular Characterization of Salmonella Phage Wara Isolated from River Water in Brazil.

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture.

Comparative genomics and phylogenomics of Campylobacter unveil potential novel species and provide insights into niche segregation.

Campylobacter is a bacterial genus associated with community outbreaks and gastrointestinal symptoms. Studies on Campylobacter generally focus on specific pathogenic species such as C. coli and C. jejuni. Currently, there are thousands of publicly available Campylobacter genomes, allowing a more complete assessment of the genus diversity. In this work, we report a network-based analysis of all available Campylobacter genomes to explore the genus structure and diversity, revealing potentially new species and elucidating genus features. We also hypothesize that the previously established Clade III of C. coli is in fact a novel species (referred here as Campylobacter spp12). Finally, we found a negative correlation between pangenome fluidity and saturation coefficient, with potential implications to the lifestyles of distinct Campylobacter species. Since pangenome analysis depends on the number of available genomes, this correlation could help estimate pangenome metrics of Campylobacter species with less sequenced genomes, helping understand their lifestyle and niche adaptation. Together, our results indicate that the Campylobacter genus should be re-evaluated, with particular attention to the interplay between genome structure and niche segregation.

Discovering and prioritizing candidate resistance genes against soybean pests by integrating GWAS and gene coexpression networks.

Soybean is one of the most important legume crops worldwide. Soybean pests have a considerable impact on crop yield. Here, we integrated publicly available genome-wide association studies and transcriptomic data to prioritize candidate resistance genes against the insects Aphis glycines and Spodoptera litura, and the nematode Heterodera glycines. We identified 171, 7, and 228 high-confidence candidate resistance genes against A. glycines, S. litura, and H. glycines, respectively. We found some overlap of candidate genes between insect species, but not between insects and H. glycines. Although 15% of the prioritized candidate genes encode proteins of unknown function, the vast majority of the candidates are related to plant immunity processes, such as transcriptional regulation, signaling, oxidative stress, recognition, and physical defense. Based on the number of resistance alleles, we selected the ten most promising accessions against each pest species in the soybean USDA germplasm. The most resistant accessions do not reach the maximum theoretical resistance potential, indicating that they might be further improved to increase resistance in breeding programs or through genetic engineering. Finally, the coexpression networks we inferred in this work are available in a user-friendly web application (https://soypestgcn.venanciogroup.uenf.br/) and an R/Shiny package (https://github.com/almeidasilvaf/SoyPestGCN) that serve as a public resource to explore soybean-pest interactions at the transcriptional level.

Network analysis of ten thousand genomes shed light on Pseudomonas diversity and classification.

The growth of sequenced bacterial genomes has revolutionized the assessment of microbial diversity. Pseudomonas is a widely diverse genus, containing more than 254 species. Although type strains have been employed to estimate Pseudomonas diversity, they represent a small fraction of the genomic diversity at a genus level. We used 10,035 available Pseudomonas genomes, including 210 type strains, to build a genomic distance network to estimate the number of species through community identification. We identified taxonomic inconsistencies with several type strains and found that 25.65 % of the Pseudomonas genomes deposited on Genbank are misclassified. The phylogenetic tree using single-copy genes from representative genomes in each species cluster in the distance network revealed at least 14 Pseudomonas groups, including the P. alcaligenes group proposed here. We show that Pseudomonas is likely an admixture of different genera and should be further divided. This study provides an overview of Pseudomonas diversity from a network and phylogenomic perspective that may help reduce the propagation of mislabeled Pseudomonas genomes.

Hybrid Genomic Analysis of Salmonella enterica Serovar Enteritidis SE3 Isolated from Polluted Soil in Brazil.

In Brazil, Salmonella enterica serovar Enteritidis is a significant health threat. Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This approach yielded 10 contigs with 99.98% identity with S. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven phage defense systems, seven prophages and a heavy metal resistance gene were encoded in the genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3, revealed an open pangenome, with a core genome of 2137 genes. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. This approach enabled the identification of key resistance and virulence genes, and these data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil.

Integrating omics approaches to discover and prioritize candidate genes involved in oil biosynthesis in soybean.

Soybean is a major source of edible protein and oil. Oil content is a quantitative trait that is significantly determined by genetic and environmental factors. Over the past 30 years, a large volume of soybean genetic, genomic, and transcriptomic data have been accumulated. Nevertheless, integrative analyses of such data remain scarce, in spite of their importance for crop improvement. We hypothesized that the co-occurrence of genomic regions for oil-related traits in different studies may reveal more stable regions encompassing important genetic determinants of oil content and quality in soybean. We integrated publicly available data, obtained with distinct techniques, to discover and prioritize candidate genes involved in oil biosynthesis and regulation in soybean. We detected key fatty acid biosynthesis genes (e.g., BCCP2 and ACCase, FADs, KAS family proteins) and several transcription factors, which are likely regulators of oil biosynthesis. In addition, we identified new candidates for seed oil accumulation and quality, such as Glyma.03G213300 and Glyma.19G160700, which encode a translocator protein homolog and a histone acetyltransferase, respectively. Further, oil and protein genomic hotspots are strongly associated with breeding and not with domestication, suggesting that soybean domestication prioritized other traits. The genes identified here are promising targets for breeding programs and for the development of soybean lines with increased oil content and quality.

BioNERO: an all-in-one R/Bioconductor package for comprehensive and easy biological network reconstruction.

Currently, standard network analysis workflows rely on many different packages, often requiring users to have a solid statistics and programming background. Here, we present BioNERO, an R package that aims to integrate all aspects of network analysis workflows, including expression data preprocessing, gene coexpression and regulatory network inference, functional analyses, and intraspecies and interspecies network comparisons. The state-of-the-art methods implemented in BioNERO ensure that users can perform all analyses with a single package in a simple pipeline, without needing to learn a myriad of package-specific syntaxes. BioNERO offers a user-friendly framework that can be easily incorporated in systems biology pipelines.

Pathogenesis-related protein 1 (PR-1) genes in soybean: Genome-wide identification, structural analysis and expression profiling under multiple biotic and abiotic stresses.

Plant pathogenesis-related (PR) proteins are a large group of proteins, classified in 17 families, that are induced by pathological conditions. Here, we characterized the soybean PR-1 (GmPR-1) gene repertoire at the sequence, structural and expression levels. We found 24 GmPR-1 genes, clustered in two phylogenetic groups. GmPR-1 genes are under strong purifying selection, particularly those that emerged by tandem duplications. GmPR-1 promoter regions are abundant in cis-regulatory elements associated with major stress-related transcription factor families, namely WRKY, ERF, HD-Zip, C2H2, NAC, and GATA. We observed that 23 GmPR-1 genes are induced by stress conditions or exclusively expressed upon stress. We explored 1972 transcriptome samples, including 26 stress conditions, revealing that most GmPR-1 genes are differentially expressed in a plethora of biotic and abiotic stresses. Our findings highlight stress-responsive GmPR-1 genes with potential biotechnological applications, such as the development of transgenic lines with increased resistance to biotic and abiotic stresses.

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus.

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

Genome sequencing of the vermicompost strain Stenotrophomonas maltophilia UENF-4GII and population structure analysis of the S. maltophilia Sm3 genogroup.

The Stenotrophomonas maltophilia complex (Smc) is a cosmopolitan bacterial group that has been proposed an emergent multidrug-resistant pathogen. Taxonomic studies support the genomic heterogeneity of Smc, which comprises genogroups exhibiting a range of phenotypically distinct strains from different sources. Here, we report the genome sequencing and in-depth analysis of S. maltophilia UENF-4GII, isolated from vermicompost. This genome harbors a unique region encoding a penicillin-binding protein (pbpX) that was carried by a transposon, as well as horizontally-transferred genomic islands involved in anti-phage defense via DNA modification, and pili glycosylation. We also analyzed all available Smc genomes to investigate genes associated with resistance and virulence, niche occupation, and population structure. S. maltophilia UENF-4GII belongs to genogroup 3 (Sm3), which comprises three phylogenetic clusters (PC). Pan-GWAS analysis uncovered 471 environment-associated and 791 PC-associated genes, including antimicrobial resistance (e.g. blaL1 and blaR1) and virulence determinants (e.g. treS and katG) that provide insights on the resistance and virulence potential of Sm3 strains. Together, the results presented here provide the grounds for more detailed clinical and ecological investigations of S. maltophilia.

Integration of genome-wide association studies and gene coexpression networks unveils promising soybean resistance genes against five common fungal pathogens.

Soybean is one of the most important legume crops worldwide. However, soybean yield is dramatically affected by fungal diseases, leading to economic losses of billions of dollars yearly. Here, we integrated publicly available genome-wide association studies and transcriptomic data to prioritize candidate genes associated with resistance to Cadophora gregata, Fusarium graminearum, Fusarium virguliforme, Macrophomina phaseolina, and Phakopsora pachyrhizi. We identified 188, 56, 11, 8, and 3 high-confidence candidates for resistance to F. virguliforme, F. graminearum, C. gregata, M. phaseolina and P. pachyrhizi, respectively. The prioritized candidate genes are highly conserved in the pangenome of cultivated soybeans and are heavily biased towards fungal species-specific defense responses. The vast majority of the prioritized candidate resistance genes are related to plant immunity processes, such as recognition, signaling, oxidative stress, systemic acquired resistance, and physical defense. Based on the number of resistance alleles, we selected the five most resistant accessions against each fungal species in the soybean USDA germplasm. Interestingly, the most resistant accessions do not reach the maximum theoretical resistance potential. Hence, they can be further improved to increase resistance in breeding programs or through genetic engineering. Finally, the coexpression network generated here is available in a user-friendly web application ( https://soyfungigcn.venanciogroup.uenf.br/ ) and an R/Shiny package ( https://github.com/almeidasilvaf/SoyFungiGCN ) that serve as a public resource to explore soybean-pathogenic fungi interactions at the transcriptional level.

Phylogenetic analysis and population structure of Pseudomonas alloputida.

The Pseudomonas putida group comprises strains with biotechnological and clinical relevance. P. alloputida was proposed as a new species and highlighted the misclassification of P. putida. Nevertheless, the population structure of P. alloputida remained unexplored. We retrieved 11,025 Pseudomonas genomes and used P. alloputida Kh7T to delineate the species. The P. alloputida population structure comprises at least 7 clonal complexes (CCs). Clinical isolates are mainly found in CC4 and acquired resistance genes are present at low frequency in plasmids. Virulence profiles support the potential of CC7 members to outcompete other plant or human pathogens through a type VI secretion system. Finally, we found that horizontal gene transfer had an important role in shaping the ability of P. alloputida to bioremediate aromatic compounds such as toluene. Our results provide the grounds to understand P. alloputida genetic diversity and its potential for biotechnological applications.

Genome-Wide Analysis of the COBRA-Like Gene Family Supports Gene Expansion through Whole-Genome Duplication in Soybean (Glycine max).

The COBRA-like (COBL) gene family has been associated with the regulation of cell wall expansion and cellulose deposition. COBL mutants result in reduced levels and disorganized deposition of cellulose causing defects in the cell wall and inhibiting plant development. In this study, we report the identification of 24 COBL genes (GmCOBL) in the soybean genome. Phylogenetic analysis revealed that the COBL proteins are divided into two groups, which differ by about 170 amino acids in the N-terminal region. The GmCOBL genes were heterogeneously distributed in 14 of the 20 soybean chromosomes. This study showed that segmental duplication has contributed significantly to the expansion of the COBL family in soybean during all Glycine-specific whole-genome duplication events. The expression profile revealed that the expression of the paralogous genes is highly variable between organs and tissues of the plant. Only 20% of the paralogous gene pairs showed similar expression patterns. The high expression levels of some GmCOBLs suggest they are likely essential for regulating cell expansion during the whole soybean life cycle. Our comprehensive overview of the COBL gene family in soybean provides useful information for further understanding the evolution and diversification of COBL genes in soybean.

Exploring the complexity of soybean (Glycine max) transcriptional regulation using global gene co-expression networks.

MAIN CONCLUSION: We report a soybean gene co-expression network built with data from 1284 RNA-Seq experiments, which was used to identify important regulators, modules and to elucidate the fates of gene duplicates. Soybean (Glycine max (L.) Merr.) is one of the most important crops worldwide, constituting a major source of protein and edible oil. Gene co-expression networks (GCN) have been extensively used to study transcriptional regulation and evolution of genes and genomes. Here, we report a soybean GCN using 1284 publicly available RNA-Seq samples from 15 distinct tissues. We found modules that are differentially regulated in specific tissues, comprising processes such as photosynthesis, gluconeogenesis, lignin metabolism, and response to biotic stress. We identified transcription factors among intramodular hubs, which probably integrate different pathways and shape the transcriptional landscape in different conditions. The top hubs for each module tend to encode proteins with critical roles, such as succinate dehydrogenase and RNA polymerase subunits. Importantly, gene essentiality was strongly correlated with degree centrality and essential hubs were enriched in genes involved in nucleic acids metabolism and regulation of cell replication. Using a guilt-by-association approach, we predicted functions for 93 of 106 hubs without functional description in soybean. Most of the duplicated genes had different transcriptional profiles, supporting their functional divergence, although paralogs originating from whole-genome duplications (WGD) are more often preserved in the same module than those from other mechanisms. Together, our results highlight the importance of GCN analysis in unraveling key functional aspects of the soybean genome, in particular those associated with hub genes and WGD events.

Systematic analysis of 1298 RNA-Seq samples and construction of a comprehensive soybean (Glycine max) expression atlas.

Soybean (Glycine max [L.] Merr.) is a major crop in animal feed and human nutrition, mainly for its rich protein and oil contents. The remarkable rise in soybean transcriptome studies over the past 5 years generated an enormous amount of RNA-seq data, encompassing various tissues, developmental conditions and genotypes. In this study, we have collected data from 1298 publicly available soybean transcriptome samples, processed the raw sequencing reads and mapped them to the soybean reference genome in a systematic fashion. We found that 94% of the annotated genes (52 737/56 044) had detectable expression in at least one sample. Unsupervised clustering revealed three major groups, comprising samples from aerial, underground and seed/seed-related parts. We found 452 genes with uniform and constant expression levels, supporting their roles as housekeeping genes. On the other hand, 1349 genes showed heavily biased expression patterns towards particular tissues. A transcript-level analysis revealed that 95% (70 963 of 74 490) of the assembled transcripts have intron chains exactly matching those from known transcripts, whereas 3256 assembled transcripts represent potentially novel splicing isoforms. The dataset compiled here constitute a new resource for the community, which can be downloaded or accessed through a user-friendly web interface at http://venanciogroup.uenf.br/resources/. This comprehensive transcriptome atlas will likely accelerate research on soybean genetics and genomics.

Polyploidization events shaped the transcription factor repertoires in legumes (Fabaceae).

Transcription factors (TFs) are essential for plant growth and development. Several legumes (e.g. soybean) are rich sources of protein and oil and have great economic importance. Here we report a phylogenomic analysis of TF families in legumes and their potential association with important traits (e.g. nitrogen fixation). We used TF DNA-binding domains to systematically screen the genomes of 15 leguminous and five non-leguminous species. Transcription factor orthologous groups (OGs) were used to estimate OG sizes in ancestral nodes using a gene birth-death model, which allowed the identification of lineage-specific expansions. The OG analysis and rate of synonymous substitutions show that major TF expansions are strongly associated with whole-genome duplication (WGD) events in the legume (approximately 58 million years ago) and Glycine (approximately 13 million years ago) lineages, which account for a large fraction of the Phaseolus vulgaris and Glycine max TF repertoires. Of the 3407 G. max TFs, 1808 and 676 have homeologs within single syntenic regions in Phaseolus vulgaris and Vitis vinifera, respectively. We found a trend for TFs expanded in legumes to be preferentially transcribed in roots and nodules, supporting their recruitment early in the evolution of nodulation in the legume clade. Some families also showed count differences between G. max and the wild soybean Glycine soja, including genes located within important quantitative trait loci. Our findings strongly support the roles of two WGDs in shaping the TF repertoires in the legume and Glycine lineages, and these are probably related to important aspects of legume and soybean biology.

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum ß-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.

Cell wall dynamics and gene expression on soybean embryonic axes during germination.

MAIN CONCLUSION: Identification of the structural changes and cell wall-related genes likely involved in cell wall extension, cellular water balance and cell wall biosynthesis on embryonic axes during germination of soybean seeds. Cell wall is a highly organized and dynamic structure that provides mechanical support for the cell. During seed germination, the cell wall is critical for cell growth and seedling establishment. Although seed germination has been widely studied in several species, key aspects regarding the regulation of cell wall dynamics in germinating embryonic axes remain obscure. Here, we characterize the gene expression patterns of cell wall pathways and investigate their impact on the cell wall dynamics of embryonic axes of germinating soybean seeds. We found 2143 genes involved in cell wall biosynthesis and assembly in the soybean genome. Key cell wall genes were highly expressed at specific germination stages, such as expansins, UDP-Glc epimerases, GT family, cellulose synthases, peroxidases, arabinogalactans, and xyloglucans-related genes. Further, we found that embryonic axes grow through modulation of these specific cell wall genes with no increment in biomass. Cell wall structural analysis revealed a defined pattern of cell expansion and an increase in cellulose content during germination. In addition, we found a clear correlation between these structural changes and expression patterns of cell wall genes during germination. Taken together, our results provide a better understanding of the complex transcriptional regulation of cell wall genes that drive embryonic axes growth and expansion during soybean germination.