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July 7, 2019  |  

Drug resistance analysis by next generation sequencing in Leishmania.

The use of next generation sequencing has the power to expedite the identification of drug resistance determinants and biomarkers and was applied successfully to drug resistance studies in Leishmania. This allowed the identification of modulation in gene expression, gene dosage alterations, changes in chromosome copy numbers and single nucleotide polymorphisms that correlated with resistance in Leishmania strains derived from the laboratory and from the field. An impressive heterogeneity at the population level was also observed, individual clones within populations often differing in both genotypes and phenotypes, hence complicating the elucidation of resistance mechanisms. This review summarizes the most recent highlights that whole genome sequencing brought to our understanding of Leishmania drug resistance and likely new directions.

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July 7, 2019  |  

The draft genome of Primula veris yields insights into the molecular basis of heterostyly.

The flowering plant Primula veris is a common spring blooming perennial that is widely cultivated throughout Europe. This species is an established model system in the study of the genetics, evolution, and ecology of heterostylous floral polymorphisms. Despite the long history of research focused on this and related species, the continued development of this system has been restricted due the absence of genomic and transcriptomic resources.We present here a de novo draft genome assembly of P. veris covering 301.8 Mb, or approximately 63% of the estimated 479.22 Mb genome, with an N50 contig size of 9.5 Kb, an N50 scaffold size of 164 Kb, and containing an estimated 19,507 genes. The results of a RADseq bulk segregant analysis allow for the confident identification of four genome scaffolds that are linked to the P. veris S-locus. RNAseq data from both P. veris and the closely related species P. vulgaris allow for the characterization of 113 candidate heterostyly genes that show significant floral morph-specific differential expression. One candidate gene of particular interest is a duplicated GLOBOSA homolog that may be unique to Primula (PveGLO2), and is completely silenced in L-morph flowers.The P. veris genome represents the first genome assembled from a heterostylous species, and thus provides an immensely important resource for future studies focused on the evolution and genetic dissection of heterostyly. As the first genome assembled from the Primulaceae, the P. veris genome will also facilitate the expanded application of phylogenomic methods in this diverse family and the eudicots as a whole.

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July 7, 2019  |  

A novel type pathway-specific regulator and dynamic genome environments of solanapyrone biosynthesis gene cluster in the fungus Ascochyta rabiei.

Secondary metabolite genes are often clustered together and situated in particular genomic regions, like the subtelomere, that can facilitate niche adaptation in fungi. Solanapyrones are toxic secondary metabolites produced by fungi occupying different ecological niches. Full-genome sequencing of the ascomycete Ascochyta rabiei revealed a solanapyrone biosynthesis gene cluster embedded in an AT-rich region proximal to a telomere end and surrounded by Tc1/Mariner-type transposable elements. The highly AT-rich environment of the solanapyrone cluster is likely the product of repeat-induced point mutations. Several secondary metabolism-related genes were found in the flanking regions of the solanapyrone cluster. Although the solanapyrone cluster appears to be resistant to repeat-induced point mutations, a P450 monooxygenase gene adjacent to the cluster has been degraded by such mutations. Among the six solanapyrone cluster genes (sol1 to sol6), sol4 encodes a novel type of Zn(II)2Cys6 zinc cluster transcription factor. Deletion of sol4 resulted in the complete loss of solanapyrone production but did not compromise growth, sporulation, or virulence. Gene expression studies with the sol4 deletion and sol4-overexpressing mutants delimited the boundaries of the solanapyrone gene cluster and revealed that sol4 is likely a specific regulator of solanapyrone biosynthesis and appears to be necessary and sufficient for induction of the solanapyrone cluster genes. Despite the dynamic surrounding genomic regions, the solanapyrone gene cluster has maintained its integrity, suggesting important roles of solanapyrones in fungal biology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Unique transposon landscapes are pervasive across Drosophila melanogaster genomes.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019  |  

Botrytis, the good, the bad and the ugly

Botrytis spp. are efficient pathogens, causing devastating diseases and significant crop losses in a wide variety of plant species. Here we outline our review of these pathogens, as well as highlight the major advances of the past 10 years in studying Botrytis in interaction with its hosts. Progress in molecular genetics and the development of relevant phylogenetic markers in particular, has resulted in the characterisation of approximately 30 species. The host range of Botrytis spp. includes plant species that are members of 170 families of cultivated plants.

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July 7, 2019  |  

Genomic insights into the taxonomic status of the three subspecies of Bacillus subtilis.

Bacillus subtilis contains three subspecies, i.e., subspecies subtilis, spizizenii, and inaquosorum. As these subspecies are phenotypically indistinguishable, their differentiation has relied on phylogenetic analysis of multiple protein-coding gene sequences. B. subtilis subsp. inaquosorum is a recently proposed taxon that encompasses strain KCTC 13429(T) and related strains, which were previously classified as members of subspecies spizizenii. However, DNA-DNA hybridization (DDH) values among the three subspecies raised a question as to their independence. Thus, we evaluated the taxonomic status of subspecies inaquosorum using genome-based comparative analysis. In contrast to the previous experimental values of DDH, the inter-genomic relatedness inferred by average nucleotide identity (ANI) values indicated that subspecies inaquosorum and spizizenii were sufficiently different from subspecies subtilis and hence raised the possibility that the former two could be classified as separate species from B. subtilis. The genome-based tree also supported the separation of the two subspecies from B. subtilis. The exclusive presence of a subtilin synthesis system in subspecies spizizenii was a remarkable genetic characteristic that could even distinguish subspecies spizizenii from subspecies inaquosorum in addition to the low ANI values (<95%). Conclusively, the genome-based data obtained in this study demonstrated that subspecies inaquosorum and spizizenii are clearly distinguished from subspecies subtilis, and raises the possibility that these two subspecies could be classified as separate species from B. subtilis. In addition, the low ANI values between subspecies inaquosorum and spizizenii and the exclusive presence of subtilin synthesis genes in subspecies spizizenii also suggest circumscription of these two subspecies at the species level. Copyright © 2013 Elsevier GmbH. All rights reserved.

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July 7, 2019  |  

Draft genome sequence of the pathogenic fungus Scedosporium apiospermum.

The first genome of one species of the Scedosporium apiospermum complex, responsible for localized to severe disseminated infections according to the immune status of the host, will contribute to a better understanding of the pathogenicity of these fungi and also to the discovery of the mechanisms underlying their low susceptibility to current antifungals. Copyright © 2014 Vandeputte et al.

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July 7, 2019  |  

Twenty-one novel microsatellite loci for the endangered Florida salt marsh vole (Microtus pennsylvanicus dukecampbelli)

We present 21 microsatellite loci developed for Florida salt marsh voles (Microtus pennsylvanicus dukecampbelli). Microsatellites were identified from single molecule real time sequencing (Pacific Biosciences). We screened 30 loci and identified 21 loci as suitable for genotyping. We screened 17 individuals from Long Cabbage Key, and 3 individuals from an unnamed island. There was no significant departure from Hardy–Weinberg equilibrium or linkage equilibrium. Fifteen of the 21 loci were variable, with overall observed heterozygosity averaging 0.39, and a mean number of alleles of 3.14. Linkage disequilibrium estimate of Ne was 10.7 (95 % CI 6.1–20.1). These markers will be useful for conservation genetics studies of this endangered species.

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July 7, 2019  |  

Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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July 7, 2019  |  

The value of new genome references.

Genomic information has become a ubiquitous and almost essential aspect of biological research. Over the last 10-15 years, the cost of generating sequence data from DNA or RNA samples has dramatically declined and our ability to interpret those data increased just as remarkably. Although it is still possible for biologists to conduct interesting and valuable research on species for which genomic data are not available, the impact of having access to a high quality whole genome reference assembly for a given species is nothing short of transformational. Research on a species for which we have no DNA or RNA sequence data is restricted in fundamental ways. In contrast, even access to an initial draft quality genome (see below for definitions) opens a wide range of opportunities that are simply not available without that reference genome assembly. Although a complete discussion of the impact of genome sequencing and assembly is beyond the scope of this short paper, the goal of this review is to summarize the most common and highest impact contributions that whole genome sequencing and assembly has had on comparative and evolutionary biology. Copyright © 2016. Published by Elsevier Inc.

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July 7, 2019  |  

Complete genome sequences of six copper-resistant Xanthomonas strains causing bacterial spot of solaneous plants, belonging to X. gardneri, X. euvesicatoria, and X. vesicatoria, using long-read technology.

Xanthomonas vesicatoria, Xanthomonas euvesicatoria, and Xanthomonas gardneri cause bacterial spot disease. Copper has been applied since the 1920s as part of integrated management programs. The first copper-resistant strains were reported some decades later. Here, we fully sequenced six Xanthomonas strains pathogenic to tomato and/or pepper and having a copper-resistant phenotype. Copyright © 2017 Richard et al.

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July 7, 2019  |  

Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses.

Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries.To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS).Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade.C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019  |  

Combination of short-read, long-read and optical mapping assemblies reveals large-scale tandem repeat arrays with population genetic implications.

Accurate and contiguous genome assembly is key to a comprehensive understanding of the processes shaping genomic diversity and evolution. Yet, it is frequently constrained by constitutive heterochromatin, usually characterized by highly repetitive DNA. As a key feature of genome architecture associated with centromeric and telomeric regions it influences meiotic recombination. In this study, we assess the impact of large tandem repeat arrays on the recombination rate landscape in an avian speciation model, the Eurasian crow. We assembled two high-quality genome references using single-molecule real-time sequencing (long-read assembly, LR) and single-molecule restriction maps (optical map assembly, OM). A three-way comparison including the published short-read assembly (SR) constructed for the same individual allowed assessing assembly properties and pinpointing mis-assemblies. Combining information from all three assemblies, we characterized 36 previously unidentified large repetitive regions in the proximity of sequence assembly breakpoints, the majority of which contained complex arrays of a 14-kb satellite repeat or its 1.2-kb subunit. Using genome-wide population re-sequencing data, we estimated the population-scaled recombination rate (?) and found it to be significantly reduced in these regions. These findings are consistent with an effect of low recombination in regions adjacent to centromeric or subtelomeric heterochromatin, and add to our understanding of the processes generating widespread heterogeneity in genetic diversity and differentiation along the genome. By combining three independent technologies, our results highlight the importance of adding a layer of information on genome structure inaccessible to each approach independently. Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019  |  

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.© 2017 Lee et al.; Published by Cold Spring Harbor Laboratory Press.

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