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April 21, 2020  |  

Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

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April 21, 2020  |  

Genomic characterization of Nocardia seriolae strains isolated from diseased fish.

Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK-14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

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April 21, 2020  |  

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.

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April 21, 2020  |  

Polysaccharide utilization loci of North Sea Flavobacteriia as basis for using SusC/D-protein expression for predicting major phytoplankton glycans.

Marine algae convert a substantial fraction of fixed carbon dioxide into various polysaccharides. Flavobacteriia that are specialized on algal polysaccharide degradation feature genomic clusters termed polysaccharide utilization loci (PULs). As knowledge on extant PUL diversity is sparse, we sequenced the genomes of 53 North Sea Flavobacteriia and obtained 400 PULs. Bioinformatic PUL annotations suggest usage of a large array of polysaccharides, including laminarin, a-glucans, and alginate as well as mannose-, fucose-, and xylose-rich substrates. Many of the PULs exhibit new genetic architectures and suggest substrates rarely described for marine environments. The isolates’ PUL repertoires often differed considerably within genera, corroborating ecological niche-associated glycan partitioning. Polysaccharide uptake in Flavobacteriia is mediated by SusCD-like transporter complexes. Respective protein trees revealed clustering according to polysaccharide specificities predicted by PUL annotations. Using the trees, we analyzed expression of SusC/D homologs in multiyear phytoplankton bloom-associated metaproteomes and found indications for profound changes in microbial utilization of laminarin, a-glucans, ß-mannan, and sulfated xylan. We hence suggest the suitability of SusC/D-like transporter protein expression within heterotrophic bacteria as a proxy for the temporal utilization of discrete polysaccharides.

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April 21, 2020  |  

Iso-Seq Allows Genome-Independent Transcriptome Profiling of Grape Berry Development.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species’ entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or “private” gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.Copyright © 2019 Minio et al.

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April 21, 2020  |  

Genetic variation in the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain.

Bacteria harboring conjugative plasmids have the potential for spreading antibiotic resistance through horizontal gene transfer. It is described that the selection and dissemination of antibiotic resistance is enhanced by stressors, like metals or antibiotics, which can occur as environmental contaminants. This study aimed at unveiling the composition of the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain (H1FC54) under different mating conditions. To meet this objective, plasmid pulsed field gel electrophoresis, optical mapping analyses and DNA sequencing were used in combination with phenotype analysis. Strain H1FC54 was observed to harbor five plasmids, three of which were conjugative and two of these, pH1FC54_330 and pH1FC54_140, contained metal and antibiotic resistance genes. Transconjugants obtained in the absence or presence of tellurite (0.5?µM or 5?µM), arsenite (0.5?µM, 5?µM or 15?µM) or ceftazidime (10?mg/L) and selected in the presence of sodium azide (100?mg/L) and tetracycline (16?mg/L) presented distinct phenotypes, associated with the acquisition of different plasmid combinations, including two co-integrate plasmids, of 310 kbp and 517 kbp. The variable composition of the conjugative plasmidome, the formation of co-integrates during conjugation, as well as the transfer of non-transferable plasmids via co-integration, and the possible association between antibiotic, arsenite and tellurite tolerance was demonstrated. These evidences bring interesting insights into the comprehension of the molecular and physiological mechanisms that underlie antibiotic resistance propagation in the environment. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

The Genome of Armadillidium vulgare (Crustacea, Isopoda) Provides Insights into Sex Chromosome Evolution in the Context of Cytoplasmic Sex Determination.

The terrestrial isopod Armadillidium vulgare is an original model to study the evolution of sex determination and symbiosis in animals. Its sex can be determined by ZW sex chromosomes, or by feminizing Wolbachia bacterial endosymbionts. Here, we report the sequence and analysis of the ZW female genome of A. vulgare. A distinguishing feature of the 1.72 gigabase assembly is the abundance of repeats (68% of the genome). We show that the Z and W sex chromosomes are essentially undifferentiated at the molecular level and the W-specific region is extremely small (at most several hundreds of kilobases). Our results suggest that recombination suppression has not spread very far from the sex-determining locus, if at all. This is consistent with A. vulgare possessing evolutionarily young sex chromosomes. We characterized multiple Wolbachia nuclear inserts in the A. vulgare genome, none of which is associated with the W-specific region. We also identified several candidate genes that may be involved in the sex determination or sexual differentiation pathways. The A. vulgare genome serves as a resource for studying the biology and evolution of crustaceans, one of the most speciose and emblematic metazoan groups. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Complete Genome Sequence of the Wolbachia wAlbB Endosymbiont of Aedes albopictus.

Wolbachia, an alpha-proteobacterium closely related to Rickettsia, is a maternally transmitted, intracellular symbiont of arthropods and nematodes. Aedes albopictus mosquitoes are naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Ae. albopictus embryos retains only wAlbB and is a key model to study host-endosymbiont interactions. We have assembled the complete circular genome of wAlbB from the Aa23 cell line using long-read PacBio sequencing at 500× median coverage. The assembled circular chromosome is 1.48 megabases in size, an increase of more than 300 kb over the published draft wAlbB genome. The annotation of the genome identified 1,205 protein coding genes, 34 tRNA, 3 rRNA, 1 tmRNA, and 3 other ncRNA loci. The long reads enabled sequencing over complex repeat regions which are difficult to resolve with short-read sequencing. Thirteen percent of the genome comprised insertion sequence elements distributed throughout the genome, some of which cause pseudogenization. Prophage WO genes encoding some essential components of phage particle assembly are missing, while the remainder are found in five prophage regions/WO-like islands or scattered around the genome. Orthology analysis identified a core proteome of 535 orthogroups across all completed Wolbachia genomes. The majority of proteins could be annotated using Pfam and eggNOG analyses, including ankyrins and components of the Type IV secretion system. KEGG analysis revealed the absence of five genes in wAlbB which are present in other Wolbachia. The availability of a complete circular chromosome from wAlbB will enable further biochemical, molecular, and genetic analyses on this strain and related Wolbachia. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Genetic Variation, Comparative Genomics, and the Diagnosis of Disease.

The discovery of mutations associated with human genetic dis- ease is an exercise in comparative genomics (see Glossary). Although there are many different strategies and approaches, the central premise is that affected persons harbor a significant excess of pathogenic DNA variants as com- pared with a group of unaffected persons (controls) that is either clinically defined1 or established by surveying large swaths of the general population.2 The more exclu- sive the variant is to the disease, the greater its penetrance, the larger its effect size, and the more relevant it becomes to both disease diagnosis and future therapeutic investigation. The most popular approach used by researchers in human genetics is the case–control design, but there are others that can be used to track variants and disease in a family context or that consider the probability of different classes of mutations based on evolutionary patterns of divergence or de novo mutational change.3,4 Although the approaches may be straightforward, the discovery of patho- genic variation and its mechanism of action often is less trivial, and decades of research can be required in order to identify the variants underlying both mendelian and complex genetic traits.

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April 21, 2020  |  

Finding Nemo’s Genes: A chromosome-scale reference assembly of the genome of the orange clownfish Amphiprion percula.

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome-scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single-molecule real-time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi-C-based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein-coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Genomic sequence and copy number evolution during hybrid crop development in sunflowers.

Hybrid crops, an important part of modern agriculture, rely on the development of male and female heterotic gene pools. In sunflowers, heterotic gene pools were developed through the use of crop-wild relatives to produce cytoplasmic male sterile female and branching, fertility restoring male lines. Here, we use genomic data from a diversity panel of male, female, and open-pollinated lines to explore the genetic changes brought during modern improvement. We find the male lines have diverged most from their open-pollinated progenitors and that genetic differentiation is concentrated in chromosomes, 8, 10 and 13, due to introgressions from wild relatives. Ancestral variation from open-pollinated varieties almost universally evolved in parallel for both male and female lines suggesting little or no selection for heterotic overdominance. Furthermore, we show that gene content differs between the male and female lines and that differentiation in gene content is concentrated in high FST regions. This means that the introgressions that brought branching and fertility restoration to the male lines, brought with them different gene content from the ancestral haplotypes, including the removal of some genes. Although we find no evidence that gene complementation genomewide is responsible for heterosis between male and female lines, several of the genes that are largely absent in either the male or female lines are associated with pathogen defense, suggesting complementation may be functionally relevant for crop breeders.

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April 21, 2020  |  

Origin and evolution of the octoploid strawberry genome.

Cultivated strawberry emerged from the hybridization of two wild octoploid species, both descendants from the merger of four diploid progenitor species into a single nucleus more than 1 million years ago. Here we report a near-complete chromosome-scale assembly for cultivated octoploid strawberry (Fragaria?×?ananassa) and uncovered the origin and evolutionary processes that shaped this complex allopolyploid. We identified the extant relatives of each diploid progenitor species and provide support for the North American origin of octoploid strawberry. We examined the dynamics among the four subgenomes in octoploid strawberry and uncovered the presence of a single dominant subgenome with significantly greater gene content, gene expression abundance, and biased exchanges between homoeologous chromosomes, as compared with the other subgenomes. Pathway analysis showed that certain metabolomic and disease-resistance traits are largely controlled by the dominant subgenome. These findings and the reference genome should serve as a powerful platform for future evolutionary studies and enable molecular breeding in strawberry.

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April 21, 2020  |  

The Genome of Cucurbita argyrosperma (Silver-Seed Gourd) Reveals Faster Rates of Protein-Coding Gene and Long Noncoding RNA Turnover and Neofunctionalization within Cucurbita.

Whole-genome duplications are an important source of evolutionary novelties that change the mode and tempo at which genetic elements evolve within a genome. The Cucurbita genus experienced a whole-genome duplication around 30 million years ago, although the evolutionary dynamics of the coding and noncoding genes in this genus have not yet been scrutinized. Here, we analyzed the genomes of four Cucurbita species, including a newly assembled genome of Cucurbita argyrosperma, and compared the gene contents of these species with those of five other members of the Cucurbitaceae family to assess the evolutionary dynamics of protein-coding and long intergenic noncoding RNA (lincRNA) genes after the genome duplication. We report that Cucurbita genomes have a higher protein-coding gene birth-death rate compared with the genomes of the other members of the Cucurbitaceae family. C. argyrosperma gene families associated with pollination and transmembrane transport had significantly faster evolutionary rates. lincRNA families showed high levels of gene turnover throughout the phylogeny, and 67.7% of the lincRNA families in Cucurbita showed evidence of birth from the neofunctionalization of previously existing protein-coding genes. Collectively, our results suggest that the whole-genome duplication in Cucurbita resulted in faster rates of gene family evolution through the neofunctionalization of duplicated genes. Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life.

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.

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