GUT MICROBIOME DIVERSITY OF THREE RHINOCEROS SPECIES IN EUROPEAN ZOOS.

The wild rhinoceros populations have declined drastically in the past decades because the rhinoceros are heavily hunted for their horns. Zoological institutions aim to conserve rhinoceros populations in captivity, but one of the challenges of ex situ conservation is to provide food sources that resemble those available in the wild. Considering that the mammalian gut microbiota is a pivotal player in their host's health, the gut microbiota of rhinoceros may also play a role in the bioavailability of nutrients. Therefore, this study aims to characterize the fecal microbiome composition of grazing white rhinoceros (WR; Ceratotherium simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) as well as the browsing black rhinoceros (BR; Diceros bicornis) kept in European zoos. Over the course of 1 yr, 166 fecal samples in total were collected from 9 BR (n = 39), 10 GOHR (n = 56), and 14 WR (n = 71) from 23 zoological institutions. The bacterial composition in the samples was determined using 16S rRNA gene Illumina sequencing. The fecal microbiomes of rhinoceros clustered by species, with BR clustering more distantly from GOHR and WR. Furthermore, the data report clustering of rhinoceros microbiota according to individual rhinoceros and institutional origin, showing that zoological institutions play a significant role in shaping the gut microbiome of rhinoceros species. In addition, BR exhibit a relatively higher microbial diversity than GOHR and WR. BR seem more susceptible to microbial gut changes and appear to have a more diverse microbiome composition among individuals than GOHR and WR. These data expand on the role of gut microbes and can provide baseline data for continued efforts in rhinoceros conservation and health status.

Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities.

BACKGROUND: Diatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging. RESULTS: Here, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed. CONCLUSIONS: Our results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

Comparative transcriptomics reveals commonalities and differences in the genetic underpinnings of a floral dimorphism.

Distyly, a floral dimorphism associated with heteromorphic self-incompatibility and controlled by the S-locus supergene, evolved independently multiple times. Comparative analyses of the first transcriptome atlas for the main distyly model, Primula veris, with other distylous species produced the following findings. A set of 53 constitutively expressed genes in P. veris did not include any of the housekeeping genes commonly used to normalize gene expression in qPCR experiments. The S-locus gene CYPT acquired its role in controlling style elongation via a change in expression profile. Comparison of genes differentially expressed between floral morphs revealed that brassinosteroids and auxin are the main hormones controlling style elongation in P. veris and Fagopyrum esculentum, respectively. Furthermore, shared biochemical pathways might underlie the expression of distyly in the distantly related P. veris, F. esculentum and Turnera subulata, suggesting a degree of correspondence between evolutionary convergence at phenotypic and molecular levels. Finally, we provide the first evidence supporting the previously proposed hypothesis that distyly supergenes of distantly related species evolved via the recruitment of genes related to the phytochrome-interacting factor (PIF) signaling network. To conclude, this is the first study that discovered homologous genes involved in the control of distyly in distantly related taxa.

Phylotranscriptomics reveals the reticulate evolutionary history of a widespread diatom species complex.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

Comparative Genomics Elucidates the Origin of a Supergene Controlling Floral Heteromorphism.

Supergenes are nonrecombining genomic regions ensuring the coinheritance of multiple, coadapted genes. Despite the importance of supergenes in adaptation, little is known on how they originate. A classic example of supergene is the S locus controlling heterostyly, a floral heteromorphism occurring in 28 angiosperm families. In Primula, heterostyly is characterized by the cooccurrence of two complementary, self-incompatible floral morphs and is controlled by five genes clustered in the hemizygous, ca. 300-kb S locus. Here, we present the first chromosome-scale genome assembly of any heterostylous species, that of Primula veris (cowslip). By leveraging the high contiguity of the P. veris assembly and comparative genomic analyses, we demonstrated that the S-locus evolved via multiple, asynchronous gene duplications and independent gene translocations. Furthermore, we discovered a new whole-genome duplication in Ericales that is specific to the Primula lineage. We also propose a mechanism for the origin of S-locus hemizygosity via nonhomologous recombination involving the newly discovered two pairs of CFB genes flanking the S locus. Finally, we detected only weak signatures of degeneration in the S locus, as predicted for hemizygous supergenes. The present study provides a useful resource for future research addressing key questions on the evolution of supergenes in general and the S locus in particular: How do supergenes arise? What is the role of genome architecture in the evolution of complex adaptations? Is the molecular architecture of heterostyly supergenes across angiosperms similar to that of Primula?

Development of a reproducible small intestinal microbiota model and its integration into the SHIME®-system, a dynamic in vitro gut model.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo. This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME®-technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus, and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo, bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology.

A pseudomolecule-scale genome assembly of the liverwort Marchantia polymorpha.

Marchantia polymorpha has recently become a prime model for cellular, evo-devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule-scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high-density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map-based cloning, feasible.

On the Role of Bioinformatics and Data Science in Industrial Microbiome Applications.

Advances in sequencing and computational biology have drastically increased our capability to explore the taxonomic and functional compositions of microbial communities that play crucial roles in industrial processes. Correspondingly, commercial interest has risen for applications where microbial communities make important contributions. These include food production, probiotics, cosmetics, and enzyme discovery. Other commercial applications include software that takes the user's gut microbiome data as one of its inputs and outputs evidence-based, automated, and personalized diet recommendations for balanced blood sugar levels. These applications pose several bioinformatic and data science challenges that range from requiring strain-level resolution in community profiles to the integration of large datasets for predictive machine learning purposes. In this perspective, we provide our insights on such challenges by touching upon several industrial areas, and briefly discuss advances and future directions of bioinformatics and data science in microbiome research.

Complete Genome Sequence of Malassezia restricta CBS 7877, an Opportunist Pathogen Involved in Dandruff and Seborrheic Dermatitis.

Malassezia restricta, one of the predominant basidiomycetous yeasts present on human skin, is involved in scalp disorders. Here, we report the complete genome sequence of the lipophilic Malassezia restricta CBS 7877 strain, which will facilitate the study of the mechanisms underlying its commensal and pathogenic roles within the skin microbiome.

A Short Indel-Lacking-Resistance Gene Triggers Silencing of the Photosynthetic Machinery Components Through TYLCSV-Associated Endogenous siRNAs in Tomato.

Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts coding for transcription factors, RNA silencing core, and disease-resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genome-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus, we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems. Among them we confirmed miR6024 targeting and cleavage of RX-coiled-coil (RX-CC), nucleotide binding site (NBS), leucine-rich (LRR) mRNA. Cleavage of paralogs was associated with short indels close to the target sites, indicating a general functional significance of indels in fine-tuning gene expression in plant-virus interaction. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several 21-22 nt secondary siRNAs. These secondary siRNAs, rather than being involved in the cascade regulation of other NBS-LRR paralogs, explained cleavages of several mRNAs annotated as defence-related proteins and components of the photosynthetic machinery. Outputs of these data explain part of the phenotype plasticity in plants, including the appearance of yellowing symptoms in the viral pathosystem.

Spread of Carbapenem Resistance by Transposition and Conjugation Among Pseudomonas aeruginosa.

The emergence of carbapenem-resistant Pseudomonas aeruginosa represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among P. aeruginosa strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-ß-lactamase VIM-2 flanked by two aacA29 genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from Pseudomonas putida. Analysis of the context of the integron showed that it is present in both strains on a ∼30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible P. aeruginosa strain. A second class 1 integron containing a gene for a CARB-2 ß-lactamase flanked by an aacA4'-8 and an aadA2 gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the oprD gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among Pseudomonas aeruginosa strains.

Novel sequencing technologies to support industrial biotechnology.

Industrial biotechnology develops and applies microorganisms for the production of bioproducts and enzymes with applications ranging from food and feed ingredients and processing to bio-based chemicals, biofuels and pharmaceutical products. Next generation DNA sequencing technologies play an increasingly important role in improving and accelerating microbial strain development for existing and novel bio-products via screening, gene and pathway discovery, metabolic engineering and additional optimization and understanding of large-scale manufacturing. In this mini-review, we describe novel DNA sequencing and analysis technologies with a focus on applications to industrial strain development, enzyme discovery and microbial community analysis.

Complete Genome Sequence of Escherichia coli AS19, an Antibiotic-Sensitive Variant of E. coli Strain B REL606.

The chemically mutagenized Escherichia coli strain AS19 was isolated on the basis of its enhanced sensitivity to different antibiotics, in particular to actinomycin. The strain was later modified to study rRNA modifications that confer antibiotic resistance. Here, we present the genome sequence of the variant E. coli AS19-RrmA.

High-Resolution Screening of Viral Communities and Identification of New Pathogens in Fish Using Next-Generation Sequencing.

Discovery of viral genomes in fish has historically been based on viral enrichment, random priming, cloning, and Sanger sequencing. However, the development of next-generation sequencing has enabled the possibility to sequence the entire virome of a tissue sample. This has led to an enormous increase in discovery of new viruses. In this chapter, we describe a simple and rapid method for viral discovery in fish. The method is based on Illumina sequencing of total RNA from diseased tissue or cell culture and in silico removal of host RNA.

Shannon Entropy to Evaluate Substitution Rate Variation Among Viral Nucleotide Positions in Datasets of Viral siRNAs.

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.

Multiple Sequence Alignment.

The increasing importance of Next Generation Sequencing (NGS) techniques has highlighted the key role of multiple sequence alignment (MSA) in comparative structure and function analysis of biological sequences. MSA often leads to fundamental biological insight into sequence-structure-function relationships of nucleotide or protein sequence families. Significant advances have been achieved in this field, and many useful tools have been developed for constructing alignments, although many biological and methodological issues are still open. This chapter first provides some background information and considerations associated with MSA techniques, concentrating on the alignment of protein sequences. Then, a practical overview of currently available methods and a description of their specific advantages and limitations are given, to serve as a helpful guide or starting point for researchers who aim to construct a reliable MSA.

NCBI-compliant genome submissions: tips and tricks to save time and money.

Genome sequences nowadays play a central role in molecular biology and bioinformatics. These sequences are shared with the scientific community through sequence databases. The sequence repositories of the International Nucleotide Sequence Database Collaboration (INSDC, comprising GenBank, ENA and DDBJ) are the largest in the world. Preparing an annotated sequence in such a way that it will be accepted by the database is challenging because many validation criteria apply. In our opinion, it is an undesirable situation that researchers who want to submit their sequence need either a lot of experience or help from partners to get the job done. To save valuable time and money, we list a number of recommendations for people who want to submit an annotated genome to a sequence database, as well as for tool developers, who could help to ease the process.

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control.

The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

Genome Sequence of EU-Unauthorized Genetically Modified Bacillus subtilis Strain 2014-3557 Overproducing Riboflavin, Isolated from a Vitamin B2 80% Feed Additive.

This paper announces the genome sequence and annotation of the genetically modified (GM) Bacillus subtilis strain 2014-3557 overproducing riboflavin (vitamin B2). This GM-strain is unauthorized in the European Union. Nevertheless, it has been isolated from a lot of vitamin B2 (riboflavin) 80% feed grade imported to Europe from China.