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April 21, 2020  |  

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

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April 21, 2020  |  

Patterns of non-ARD variation in more than 300 full-length HLA-DPB1 alleles.

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation. The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A???G variant, a known expression marker located in the 3′-UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6?kb stretch at the 3′-end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles. The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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April 21, 2020  |  

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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April 21, 2020  |  

Bioinformatic analysis of the complete genome sequence of Pectobacterium carotovorum subsp. brasiliense BZA12 and candidate effector screening

AbstractPectobacterium carotovorum subsp. brasiliense (Pcb) is a gram-negative, plant pathogenic bacterium of the soft rot Enterobacteriaceae (SRE) family. We present the complete genome sequence of Pcb strain BZA12, which reveals that Pcb strain BZA12 carries a single 4,924,809 bp chromosome with 51.97% GC content and comprises 4508 predicted protein-coding genes.Geneannotationofthese genes utilizedGO, KEGG,and COG databases.Incomparison withthree closely related soft-rot pathogens, strain BZA12 has 3797 gene families, among which 3107 gene families are identified as orthologous with those of both P. carotovorum subsp. carotovorum PCC21 and P. carotovorum subsp. odoriferum BCS7, as well as 36 putative Unique Gene Families. We selected five putative effectors from the BZA12 genome and transiently expressed them in Nicotiana benthamiana. Candidate effector A12GL002483 was localized in the cell nucleus and induced cell death. This study provides a foundation for a better understanding of the genomic structure and function of Pcb, particularly in the discovery of potential pathogenic factors and for the development of more effective strategies against this pathogen.

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April 21, 2020  |  

Genome assembly and gene expression in the American black bear provides new insights into the renal response to hibernation.

The prevalence of chronic kidney disease (CKD) is rising worldwide and 10-15% of the global population currently suffers from CKD and its complications. Given the increasing prevalence of CKD there is an urgent need to find novel treatment options. The American black bear (Ursus americanus) copes with months of lowered kidney function and metabolism during hibernation without the devastating effects on metabolism and other consequences observed in humans. In a biomimetic approach to better understand kidney adaptations and physiology in hibernating black bears, we established a high-quality genome assembly. Subsequent RNA-Seq analysis of kidneys comparing gene expression profiles in black bears entering (late fall) and emerging (early spring) from hibernation identified 169 protein-coding genes that were differentially expressed. Of these, 101 genes were downregulated and 68 genes were upregulated after hibernation. Fold changes ranged from 1.8-fold downregulation (RTN4RL2) to 2.4-fold upregulation (CISH). Most notable was the upregulation of cytokine suppression genes (SOCS2, CISH, and SERPINC1) and the lack of increased expression of cytokines and genes involved in inflammation. The identification of these differences in gene expression in the black bear kidney may provide new insights in the prevention and treatment of CKD. © The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020  |  

Genome Sequence of Jaltomata Addresses Rapid Reproductive Trait Evolution and Enhances Comparative Genomics in the Hyper-Diverse Solanaceae.

Within the economically important plant family Solanaceae, Jaltomata is a rapidly evolving genus that has extensive diversity in flower size and shape, as well as fruit and nectar color, among its ~80 species. Here, we report the whole-genome sequencing, assembly, and annotation, of one representative species (Jaltomata sinuosa) from this genus. Combining PacBio long reads (25×) and Illumina short reads (148×) achieved an assembly of ~1.45?Gb, spanning ~96% of the estimated genome. Ninety-six percent of curated single-copy orthologs in plants were detected in the assembly, supporting a high level of completeness of the genome. Similar to other Solanaceous species, repetitive elements made up a large fraction (~80%) of the genome, with the most recently active element, Gypsy, expanding across the genome in the last 1-2 Myr. Computational gene prediction, in conjunction with a merged transcriptome data set from 11 tissues, identified 34,725 protein-coding genes. Comparative phylogenetic analyses with six other sequenced Solanaceae species determined that Jaltomata is most likely sister to Solanum, although a large fraction of gene trees supported a conflicting bipartition consistent with substantial introgression between Jaltomata and Capsicum after these species split. We also identified gene family dynamics specific to Jaltomata, including expansion of gene families potentially involved in novel reproductive trait development, and loss of gene families that accompanied the loss of self-incompatibility. This high-quality genome will facilitate studies of phenotypic diversification in this rapidly radiating group and provide a new point of comparison for broader analyses of genomic evolution across the Solanaceae.

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April 21, 2020  |  

Heterochromatin-enriched assemblies reveal the sequence and organization of the Drosophila melanogaster Y chromosome.

Heterochromatic regions of the genome are repeat-rich and poor in protein coding genes, and are therefore underrepresented in even the best genome assemblies. One of the most difficult regions of the genome to assemble are sex-limited chromosomes. The Drosophila melanogaster Y chromosome is entirely heterochromatic, yet has wide-ranging effects on male fertility, fitness, and genome-wide gene expression. The genetic basis of this phenotypic variation is difficult to study, in part because we do not know the detailed organization of the Y chromosome. To study Y chromosome organization in D. melanogaster, we develop an assembly strategy involving the in silico enrichment of heterochromatic long single-molecule reads and use these reads to create targeted de novo assemblies of heterochromatic sequences. We assigned contigs to the Y chromosome using Illumina reads to identify male-specific sequences. Our pipeline extends the D. melanogaster reference genome by 11.9 Mb, closes 43.8% of the gaps, and improves overall contiguity. The addition of 10.6 MB of Y-linked sequence permitted us to study the organization of repeats and genes along the Y chromosome. We detected a high rate of duplication to the pericentric regions of the Y chromosome from other regions in the genome. Most of these duplicated genes exist in multiple copies. We detail the evolutionary history of one sex-linked gene family, crystal-Stellate While the Y chromosome does not undergo crossing over, we observed high gene conversion rates within and between members of the crystal-Stellate gene family, Su(Ste), and PCKR, compared to genome-wide estimates. Our results suggest that gene conversion and gene duplication play an important role in the evolution of Y-linked genes. Copyright © 2019 Chang and Larracuente.

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April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020  |  

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii).

Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini-Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig-N50 2.2 Mb and Scaffold-N50 5.2 Mb) for F. hindsii. In total, 32 257 protein-coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR-modified F. hindsii were predominantly 1-bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast.

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ~20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity. © 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  |  

Structural and functional characterization of an intradiol ring-cleavage dioxygenase from the polyphagous spider mite herbivore Tetranychus urticae Koch.

Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 17 genes that code for secreted proteins belonging to the “intradiol dioxygenase-like” subgroup. Phylogenetic analyses indicate that this novel enzyme family has been acquired by horizontal gene transfer. In order to better understand the role of these proteins in T. urticae, we have structurally and functionally characterized one paralog (tetur07g02040). It was demonstrated that this protein is indeed an intradiol ring-cleavage dioxygenase, as the enzyme is able to cleave catechol between two hydroxyl-groups using atmospheric dioxygen. The enzyme was characterized functionally and structurally. The active site of the T. urticae enzyme contains an Fe3+ cofactor that is coordinated by two histidine and two tyrosine residues, an arrangement that is similar to those observed in bacterial homologs. However, the active site is significantly more solvent exposed than in bacterial proteins. Moreover, the mite enzyme is monomeric, while almost all structurally characterized bacterial homologs form oligomeric assemblies. Tetur07g02040 is not only the first spider mite dioxygenase that has been characterized at the molecular level, but is also the first structurally characterized intradiol ring-cleavage dioxygenase originating from a eukaryote.Copyright © 2018 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Midrib Sucrose Accumulation and Sugar Transporter Gene Expression in YCS-Affected Sugarcane Leaves

Sucrose accumulation and decreased photosynthesis are early symptoms of yellow canopy syndrome (YCS) in sugarcane (Saccharum spp.), and precede the visual yellowing of the leaves. To investigate broad-scale gene expression changes during YCS-onset, transcriptome analyses coupled to metabolome analyses were performed. Across leaf tissues, the greatest number of differentially expressed genes related to the chloroplast, and the metabolic processes relating to nitrogen and carbohydrates. Five genes represented 90% of the TPM (Transcripts Per Million) associated with the downregulation of transcription during YCS-onset, which included PSII D1 (PsbA). This differential expression was consistent with a feedback regulatory effect upon photosynthesis. Broad-scale gene expression analyses did not reveal a cause for leaf sugar accumulation during YCS-onset. Interestingly, the midrib showed the greatest accumulation of sugars, followed by symptomatic lamina. To investigate if phloem loading/reloading may be compromised on a gene expression level – to lead to leaf sucrose accumulation – sucrose transport-related proteins of SWEETs, Sucrose Transporters (SUTs), H+-ATPases and H+-pyrophosphatases (H+-PPases) were characterised from a sugarcane transcriptome and expression analysed. Two clusters of Type I H+-PPases, with one upregulated and the other downregulated, were evident. Although less pronounced, a similar pattern of change was observed for the H+-ATPases. The disaccharide transporting SWEETs were downregulated after visual symptoms were present, and a monosaccharide transporting SWEET upregulated preceding, as well as after, symptom development. SUT gene expression was the least responsive to YCS development. The results are consistent with a reduction of photoassimilate movement through the phloem leading to sucrose build-up in the leaf.

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April 21, 2020  |  

A siphonous macroalgal genome suggests convergent functions of homeobox genes in algae and land plants.

Genome evolution and development of unicellular, multinucleate macroalgae (siphonous algae) are poorly known, although various multicellular organisms have been studied extensively. To understand macroalgal developmental evolution, we assembled the ~26?Mb genome of a siphonous green alga, Caulerpa lentillifera, with high contiguity, containing 9,311 protein-coding genes. Molecular phylogeny using 107 nuclear genes indicates that the diversification of the class Ulvophyceae, including C. lentillifera, occurred before the split of the Chlorophyceae and Trebouxiophyceae. Compared with other green algae, the TALE superclass of homeobox genes, which expanded in land plants, shows a series of lineage-specific duplications in this siphonous macroalga. Plant hormone signalling components were also expanded in a lineage-specific manner. Expanded transport regulators, which show spatially different expression, suggest that the structural patterning strategy of a multinucleate cell depends on diversification of nuclear pore proteins. These results not only imply functional convergence of duplicated genes among green plants, but also provide insight into evolutionary roots of green plants. Based on the present results, we propose cellular and molecular mechanisms involved in the structural differentiation in the siphonous alga. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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