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July 7, 2019  |  

Strategies for sequence assembly of plant genomes

The field of plant genome assembly has greatly benefited from the development and widespread adoption of next-generation DNA sequencing platforms. Very high sequencing throughputs and low costs per nucleotide have considerably reduced the technical and budgetary constraints associated with early assembly projects done primarily with a traditional Sanger-based approach. Those improvements led to a sharp increase in the number of plant genomes being sequenced, including large and complex genomes of economically important crops. Although next-generation DNA sequencing has considerably improved our understanding of the overall structure and dynamics of many plant genomes, severe limitations still remain because next-generation DNA sequencing reads typically are shorter than Sanger reads. In addition, the software tools used to de novo assemble sequences are not necessarily designed to optimize the use of short reads. These cause challenges, common to many plant species with large genome sizes, high repeat contents, polyploidy and genome-wide duplications. This chapter provides an overview of historical and current methods used to sequence and assemble plant genomes, along with new solutions offered by the emergence of technologies such as single molecule sequencing and optical mapping to address the limitations of current sequence assemblies.

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July 7, 2019  |  

Chloroplast genome sequence of Arabidopsis thaliana accession Landsberg erecta, assembled from single-molecule, real-time sequencing data.

A publicly available data set from Pacific Biosciences was used to create an assembly of the chloroplast genome sequence of the Arabidopsis thaliana genotype Landsberg erecta The assembly is solely based on single-molecule, real-time sequencing data and hence provides high resolution of the two inverted repeat regions typically contained in chloroplast genomes. Copyright © 2016 Stadermann et al.

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July 7, 2019  |  

Comparative evaluation of the genomes of three common Drosophila-associated bacteria.

Drosophila melanogaster is an excellent model to explore the molecular exchanges that occur between an animal intestine and associated microbes. Previous studies in Drosophila uncovered a sophisticated web of host responses to intestinal bacteria. The outcomes of these responses define critical events in the host, such as the establishment of immune responses, access to nutrients, and the rate of larval development. Despite our steady march towards illuminating the host machinery that responds to bacterial presence in the gut, there are significant gaps in our understanding of the microbial products that influence bacterial association with a fly host. We sequenced and characterized the genomes of three common Drosophila-associated microbes: Lactobacillus plantarum, Lactobacillus brevis and Acetobacter pasteurianus For each species, we compared the genomes of Drosophila-associated strains to the genomes of strains isolated from alternative sources. We found that environmental Lactobacillus strains readily associated with adult Drosophila and were similar to fly isolates in terms of genome organization. In contrast, we identified a strain of A. pasteurianus that apparently fails to associate with adult Drosophila due to an inability to grow on fly nutrient food. Comparisons between association competent and incompetent A. pasteurianus strains identified a short list of candidate genes that may contribute to survival on fly medium. Many of the gene products unique to fly-associated strains have established roles in the stabilization of host-microbe interactions. These data add to a growing body of literature that examines the microbial perspective of host-microbe relationships. © 2016. Published by The Company of Biologists Ltd.

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July 7, 2019  |  

Mobile genetic elements: in silico, in vitro, in vivo.

Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function and evolution. However, there is currently a gap between the fast pace of TE discovery in silico, driven by the exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, ‘Mobile Genetic Elements: in silico, in vitro, in vivo’, held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.© 2016 John Wiley & Sons Ltd.

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July 7, 2019  |  

Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.

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July 7, 2019  |  

‘Candidatus Tenderia electrophaga’, an uncultivated electroautotroph from a biocathode enrichment.

Biocathode communities are of interest for a variety of applications, including electrosynthesis, bioremediation, and biosensors, yet much remains to be understood about the biological processes that occur to enable these communities to grow. One major difficulty in understanding these communities is that the critical autotrophic organisms are difficult to cultivate. An uncultivated, electroautotrophic bacterium previously identified as an uncultivated member of the family Chromatiaceae appears to be a key organism in an autotrophic biocathode microbial community. Metagenomic, metaproteomic and metatranscriptomic characterization of this community indicates that there is likely a single organism that utilizes electrons from the cathode to fix CO2, yet this organism has not been obtained in pure culture. Fluorescence in situ hybridization reveals that the organism grows as rod-shaped cells approximately 1.8 × 0.6 µm, and forms large clumps on the cathode. The genomic DNA G+C content was 59.2 mol%. Here we identify the key features of this organism and propose ‘Candidatus Tenderia electrophaga’, within the Gammaproteobacteria on the basis of low nucleotide and predicted protein sequence identity to known members of the orders Chromatiales and Thiotrichales.

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July 7, 2019  |  

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88?Mb (contig N50 of 1.87?Mb), covering 98% of the 750?Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

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July 7, 2019  |  

ChIP-Seq-annotated Heliconius erato genome highlights patterns of cis-regulatory evolution in Lepidoptera.

Uncovering phylogenetic patterns of cis-regulatory evolution remains a fundamental goal for evolutionary and developmental biology. Here, we characterize the evolution of regulatory loci in butterflies and moths using chromatin immunoprecipitation sequencing (ChIP-seq) annotation of regulatory elements across three stages of head development. In the process we provide a high-quality, functionally annotated genome assembly for the butterfly, Heliconius erato. Comparing cis-regulatory element conservation across six lepidopteran genomes, we find that regulatory sequences evolve at a pace similar to that of protein-coding regions. We also observe that elements active at multiple developmental stages are markedly more conserved than elements with stage-specific activity. Surprisingly, we also find that stage-specific proximal and distal regulatory elements evolve at nearly identical rates. Our study provides a benchmark for genome-wide patterns of regulatory element evolution in insects, and it shows that developmental timing of activity strongly predicts patterns of regulatory sequence evolution. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Probabilistic viral quasispecies assembly

Viruses are pathogens that cause infectious diseases. The swarm of virions is subject to the host’s immune pressure and possibly antiviral therapy. It may escape this selective pressure and gain selective advantage by acquiring one or more of the genomic alterations: single-nucleotide variants (SNVs), loss or gain of one or more amino acids, large deletions, for example, due to alternative splicing, or recombination of different strains. Genotypic antiretroviral drug resistance testing is performed via sequencing. Next-generation sequencing (NGS) technologies revolutionized assessing viral genetic diversity experimentally. In viral quasispecies analysis, there are two main goals: the identification of low-frequency variants and haplotype assembly on a whole-genome scale. PacBio performs single-molecule sequencing. This chapter elaborates human haplotyping and its relationship to probabilistic viral haplotype reconstruction methods. Viral quasispecies assembly has the potential to replace the current de facto diversity estimation by SNV calling. With advances in library preparation, increasing sensitivity of sequencing platforms, and more sophisticated models, it might be possible to detect all or most viral strains in a single individual.

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July 7, 2019  |  

The mechanisms whereby the green alga Chlorella ohadii, isolated from desert soil crust, exhibits unparalleled photodamage resistance.

Excess illumination damages the photosynthetic apparatus with severe implications with regard to plant productivity. Unlike model organisms, the growth of Chlorella ohadii, isolated from desert soil crust, remains unchanged and photosynthetic O2 evolution increases, even when exposed to irradiation twice that of maximal sunlight. Spectroscopic, biochemical and molecular approaches were applied to uncover the mechanisms involved. D1 protein in photosystem II (PSII) is barely degraded, even when exposed to antibiotics that prevent its replenishment. Measurements of various PSII parameters indicate that this complex functions differently from that in model organisms and suggest that C. ohadii activates a nonradiative electron recombination route which minimizes singlet oxygen formation and the resulting photoinhibition. The light-harvesting antenna is very small and carotene composition is hardly affected by excess illumination. Instead of succumbing to photodamage, C. ohadii activates additional means to dissipate excess light energy. It undergoes major structural, compositional and physiological changes, leading to a large rise in photosynthetic rate, lipids and carbohydrate content and inorganic carbon cycling. The ability of C. ohadii to avoid photodamage relies on a modified function of PSII and the dissipation of excess reductants downstream of the photosynthetic reaction centers. The biotechnological potential as a gene source for crop plant improvement is self-evident.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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July 7, 2019  |  

A full-body transcriptome and proteome resource for the European common carp.

The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies.Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data.The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

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July 7, 2019  |  

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

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July 7, 2019  |  

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.

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July 7, 2019  |  

Cell cycle constraints and environmental control of local DNA hypomethylation in a-proteobacteria.

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of a-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5′-GANTC-3′ sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different a-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.

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July 7, 2019  |  

TeloPCR-seq: a high-throughput sequencing approach for telomeres.

We have developed a high-throughput sequencing approach that enables us to determine terminal telomere sequences from tens of thousands of individual Schizosaccharomyces pombe telomeres. This method provides unprecedented coverage of telomeric sequence complexity in fission yeast. S. pombe telomeres are composed of modular degenerate repeats that can be explained by variation in usage of the TER1 RNA template during reverse transcription. Taking advantage of this deep sequencing approach, we find that ‘like’ repeat modules are highly correlated within individual telomeres. Moreover, repeat module preference varies with telomere length, suggesting that existing repeats promote the incorporation of like repeats and/or that specific conformations of the telomerase holoenzyme efficiently and/or processively add repeats of like nature. After the loss of telomerase activity, this sequencing and analysis pipeline defines a population of telomeres with altered sequence content. This approach will be adaptable to study telomeric repeats in other organisms and also to interrogate repetitive sequences throughout the genome that are inaccessible to other sequencing methods.© 2016 Federation of European Biochemical Societies.

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