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July 7, 2019

Comparative mitogenomic analysis of three species of periwinkles: Littorina fabalis, L. obtusata and L. saxatilis.

The flat periwinkles, Littorina fabalis and L. obtusata, offer an interesting system for local adaptation and ecological speciation studies. In order to provide genomic resources for these species, we sequenced their mitogenomes together with that of the rough periwinkle L. saxatilis by means of next-generation sequencing technologies. The three mitogenomes present the typical repertoire of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a putative control region. Although the latter could not be fully recovered in flat periwinkles using short-reads due to a highly repetitive fragment, in L. saxatilis this problem was overcome with additional long-reads and we were able to assemble the complete mitogenome. Both gene order and nucleotide composition are similar between the three species as well as compared to other Littorinimorpha. A large variance in divergence was observed across mitochondrial regions, with six- to ten-fold difference between the highest and the lowest divergence rates. Based on nucleotide changes on the whole molecule and assuming a molecular clock, L. fabalis and L. obtusata started to diverge around 0.8 Mya (0.4-1.1 Mya). The evolution of the mitochondrial protein-coding genes in the three Littorina species appears mainly influenced by purifying selection as revealed by phylogenetic tests based on dN/dS ratios that did not detect any evidence for positive selection, although some caution is required given the limited power of the dataset and the implemented approaches. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes.Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source.The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from the phyllosphere. Noteworthy was the strong antagonistic activity of the soil yeast C. subhashii, which had so far only been described from a clinical sample and not been studied with respect to biocontrol. Based on binary competition assays and growth analyses (e.g., on different carbon sources, growth in root exudates), C. subhashii was identified as a competitive and antagonistic soil yeast with potential as a novel biocontrol agent against plant pathogenic fungi.

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July 7, 2019

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

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July 7, 2019

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

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July 7, 2019

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephulotus folliculnris, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identi- fied genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. folliculnris and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.

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July 7, 2019

Complex modular architecture around a simple toolkit of wing pattern genes

Identifying the genomic changes that control morphological variation and understanding how they generate diversity is a major goal of evolutionary biology. In Heliconius butterflies, a small number of genes control the development of diverse wing colour patterns. Here, we used full-genome sequencing of individuals across the Heliconius erato radiation and closely related species to characterize genomic variation associated with wing pattern diversity. We show that variation around colour pattern genes is highly modular, with narrow genomic intervals associated with specific differences in colour and pattern. This modular architecture explains the diversity of colour patterns and provides a flexible mechanism for rapid morphological diversification.

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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

The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two ‘types’, G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for “dead-end” trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

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July 7, 2019

Comparative genomics and transcriptome analysis of Aspergillus niger and metabolic engineering for citrate production.

Despite a long and successful history of citrate production in Aspergillus niger, the molecular mechanism of citrate accumulation is only partially understood. In this study, we used comparative genomics and transcriptome analysis of citrate-producing strains-namely, A. niger H915-1 (citrate titer: 157?g?L(-1)), A1 (117?g?L(-1)), and L2 (76?g?L(-1))-to gain a genome-wide view of the mechanism of citrate accumulation. Compared with A. niger A1 and L2, A. niger H915-1 contained 92 mutated genes, including a succinate-semialdehyde dehydrogenase in the ?-aminobutyric acid shunt pathway and an aconitase family protein involved in citrate synthesis. Furthermore, transcriptome analysis of A. niger H915-1 revealed that the transcription levels of 479 genes changed between the cell growth stage (6?h) and the citrate synthesis stage (12?h, 24?h, 36?h, and 48?h). In the glycolysis pathway, triosephosphate isomerase was up-regulated, whereas pyruvate kinase was down-regulated. Two cytosol ATP-citrate lyases, which take part in the cycle of citrate synthesis, were up-regulated, and may coordinate with the alternative oxidases in the alternative respiratory pathway for energy balance. Finally, deletion of the oxaloacetate acetylhydrolase gene in H915-1 eliminated oxalate formation but neither influence on pH decrease nor difference in citrate production were observed.

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July 7, 2019

Genome scaffolding and annotation for the pathogen vector Ixodes ricinus by ultra-long single molecule sequencing.

Global warming and other ecological changes have facilitated the expansion of Ixodes ricinus tick populations. Ixodes ricinus is the most important carrier of vector-borne pathogens in Europe, transmitting viruses, protozoa and bacteria, in particular Borrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis, the most prevalent vector-borne disease in humans in the Northern hemisphere. To faster control this disease vector, a better understanding of the I. ricinus tick is necessary. To facilitate such studies, we recently published the first reference genome of this highly prevalent pathogen vector. Here, we further extend these studies by scaffolding and annotating the first reference genome by using ultra-long sequencing reads from third generation single molecule sequencing. In addition, we present the first genome size estimation for I. ricinus ticks and the embryo-derived cell line IRE/CTVM19.235,953 contigs were integrated into 204,904 scaffolds, extending the currently known genome lengths by more than 30% from 393 to 516 Mb and the N50 contig value by 87% from 1643 bp to a N50 scaffold value of 3067 bp. In addition, 25,263 sequences were annotated by comparison to the tick’s North American relative Ixodes scapularis. After (conserved) hypothetical proteins, zinc finger proteins, secreted proteins and P450 coding proteins were the most prevalent protein categories annotated. Interestingly, more than 50% of the amino acid sequences matching the homology threshold had 95-100% identity to the corresponding I. scapularis gene models. The sequence information was complemented by the first genome size estimation for this species. Flow cytometry-based genome size analysis revealed a haploid genome size of 2.65Gb for I. ricinus ticks and 3.80 Gb for the cell line.We present a first draft sequence map of the I. ricinus genome based on a PacBio-Illumina assembly. The I. ricinus genome was shown to be 26% (500 Mb) larger than the genome of its American relative I. scapularis. Based on the genome size of 2.65 Gb we estimated that we covered about 67% of the non-repetitive sequences. Genome annotation will facilitate screening for specific molecular pathways in I. ricinus cells and provides an overview of characteristics and functions.

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July 7, 2019

Non hybrid long read consensus using local De Bruijn graph assembly

While second generation sequencing led to a vast increase in sequenced data, the shorter reads which came with it made assembly a much harder task and for some regions impossible with only short read data. This changed again with the advent of third generation long read sequencers. The length of the long reads allows a much better resolution of repetitive regions, their high error rate however is a major challenge. Using the data successfully requires to remove most of the sequencing errors. The first hybrid correction methods used low noise second generation data to correct third generation data, but this approach has issues when it is unclear where to place the short reads due to repeats and also because second generation sequencers fail to sequence some regions which third generation sequencers work on. Later non hybrid methods appeared. We present a new method for non hybrid long read error correction based on De Bruijn graph assembly of short windows of long reads with subsequent combination of these correct windows to corrected long reads. Our experiments show that this method yields a better correction than other state of the art non hybrid correction approaches.

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July 7, 2019

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

A genomic view of short tandem repeats.

Short tandem repeats (STRs) are some of the fastest mutating loci in the genome. Tools for accurately profiling STRs from high-throughput sequencing data have enabled genome-wide interrogation of more than a million STRs across hundreds of individuals. These catalogs have revealed that STRs are highly multiallelic and may contribute more de novo mutations than any other variant class. Recent studies have leveraged these catalogs to show that STRs play a widespread role in regulating gene expression and other molecular phenotypes. These analyses suggest that STRs are an underappreciated but rich reservoir of variation that likely make significant contributions to Mendelian diseases, complex traits, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 7, 2019

Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization.

Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into “attachment discs” that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (~100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin type, while motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive “linker”, which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019

A pipeline for local assembly of minisatellite alleles from single-molecule sequencing data.

The advent of Next Generation Sequencing (NGS) has led to the generation of enormous volumes of short read sequence data, cheaply and in reasonable time scales. Nevertheless, the quality of genome assemblies generated using NGS technologies has been greatly affected, compared to those generated using Sanger DNA sequencing. This is largely due to the inability of short read sequence data to scaffold repetitive structures, creating gaps, inversions and rearrangements and resulting in assemblies that are, at best, draft forms. Third generation single-molecule sequencing (SMS) technologies (e.g. Pacific Biosciences Single Molecule Real Time (SMRT) system) address this challenge by generating sequences with increased read lengths, offering the prospect to better recover these complex repetitive structures, concomitantly improving assembly quality.Here, we evaluate the ability of SMS data (specifically human genome Pacific Biosciences SMRT data) to recover poorly represented repetitive sequences (specifically, GC-rich human minisatellites). To do this we designed a pipeline for the collection, processing and local assembly of single-molecule sequence data to form accurate contiguous local reconstructions. Our results show the recovery of an allele of the non-coding minisatellite MS1 (located on chromosome 1 at 1p33-35) at greater than 97% identity to reference (GRCh38) from the unprocessed sequence data of a haploid complete hydatidiform mole (CHM1) cell line. Furthermore, our assembly revealed an allele of over 500 repeat units; much larger than the reference (GRCh38), but consistent in structure with naturally occurring alleles that are segregating in human populations. This local assembly’s reconstruction was validated with the release of the whole genome assemblies GCA_001297185.1 and GCA_000772585.3, where this allele occurs. Additionally, application of this pipeline to coding minisatellites in the PRDM9 and ZNF93 genes enabled recovery of high identity allele structures for these sequence regions whose length was confirmed by PCR from cell line genomic DNA. The internal repeat structure of the PRDM9 allele recovered was consistent with common human-specific alleles.Code available at https://github.com/ndliberial/smrt_pipeline CONTACT: dno2@le.ac.uk. © The Author 2016. Published by Oxford University Press.

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