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September 22, 2019  |  

Draft genome of Glyptosternon maculatum, an endemic fish from Tibet Plateau.

Mechanisms for high-altitude adaption have attracted widespread interest among evolutionary biologists. Several genome-wide studies have been carried out for endemic vertebrates in Tibet, including mammals, birds, and amphibians. However, little information is available about the adaptive evolution of highland fishes. Glyptosternon maculatum (Regan 1905), also known as Regan or barkley and endemic to the Tibetan Plateau, belongs to the Sisoridae family, order Siluriformes (catfishes). This species lives at an elevation ranging from roughly 2,800 m to 4,200 m. Hence, a high-quality reference genome of G. maculatum provides an opportunity to investigate high-altitude adaption mechanisms of fishes.To obtain a high-quality reference genome sequence of G. maculatum, we combined Pacific Bioscience single-molecule real-time sequencing, Illumina paired-end sequencing, 10X Genomics linked-reads, and BioNano optical map techniques. In total, 603.99 Gb sequencing data were generated. The assembled genome was about 662.34 Mb with scaffold and contig N50 sizes of 20.90 Mb and 993.67 kb, respectively, which captured 83% complete and 3.9% partial vertebrate Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 35.88% of the genome, and ?22,066 protein-coding genes were predicted from the genome, of which 91.7% have been functionally annotated.We present the first comprehensive de novo genome of G. maculatum. This genetic resource is fundamental for investigating the origin of G. maculatum and will improve our understanding of high-altitude adaption of fishes. The assembled genome can also be used as reference for future population genetic studies of G. maculatum.

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September 22, 2019  |  

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms “Membrane” and “Protein Secretion.”In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

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September 22, 2019  |  

Draft genome sequence of wild Prunus yedoensis reveals massive inter-specific hybridization between sympatric flowering cherries.

Hybridization is an important evolutionary process that results in increased plant diversity. Flowering Prunus includes popular cherry species that are appreciated worldwide for their flowers. The ornamental characteristics were acquired both naturally and through artificially hybridizing species with heterozygous genomes. Therefore, the genome of hybrid flowering Prunus presents important challenges both in plant genomics and evolutionary biology.We use long reads to sequence and analyze the highly heterozygous genome of wild Prunus yedoensis. The genome assembly covers >?93% of the gene space; annotation identified 41,294 protein-coding genes. Comparative analysis of the genome with 16 accessions of six related taxa shows that 41% of the genes were assigned into the maternal or paternal state. This indicates that wild P. yedoensis is an F1 hybrid originating from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura, and it can be clearly distinguished from its confusing taxon, Yoshino cherry. A focused analysis of the S-locus haplotypes of closely related taxa distributed in a sympatric natural habitat suggests that reduced restriction of inter-specific hybridization due to strong gametophytic self-incompatibility is likely to promote complex hybridization of wild Prunus species and the development of a hybrid swarm.We report the draft genome assembly of a natural hybrid Prunus species using long-read sequencing and sequence phasing. Based on a comprehensive comparative genome analysis with related taxa, it appears that cross-species hybridization in sympatric habitats is an ongoing process that facilitates the diversification of flowering Prunus.

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September 22, 2019  |  

A draft genome assembly of the Chinese sillago (Sillago sinica), the first reference genome for Sillaginidae fishes.

Sillaginidae, also known as smelt-whitings, is a family of benthic coastal marine fishes in the Indo-West Pacific that have high ecological and economic importance. Many Sillaginidae species, including the Chinese sillago (Sillago sinica), have been recently described in China, providing valuable material to analyze genetic diversification of the family Sillaginidae. Here, we constructed a reference genome for the Chinese sillago, with the aim to set up a platform for comparative analysis of all species in this family.Using the single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) Sequel, we generated ~27.3 Gb genomic DNA sequences for the Chinese sillago. We reconstructed a genome assembly of 534 Mb using a strategy that takes advantage of complementary strengths of two genome assembly programs, Canu and FALCON. The genome size was consistent with the estimated genome size based on k-mer analysis. The assembled genome consisted of 802 contigs with a contig N50 length of 2.6 Mb. We annotated 22,122 protein-coding genes in the Chinese sillago genomes using a de novo method as well as RNA sequencing data and homologies to other teleosts. According to the phylogenetic analysis using protein-coding genes, the Chinese sillago is closely related to Larimichthys crocea and Dicentrarchus labrax and diverged from their ancestor around 69.5-82.6 million years ago.Using long reads generated with PacBio sequencing technology, we have built a draft genome assembly for the Chinese sillago, which is the first reference genome for Sillaginidae species. This genome assembly sets a stage for comparative analysis of the diversification and adaptation of fishes in Sillaginidae.

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September 22, 2019  |  

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019  |  

4.5 years within-patient evolution of a colistin resistant KPC-producing Klebsiella pneumoniae ST258.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown.We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated.The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp.Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

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September 22, 2019  |  

Genome sequence of the cauliflower mushroom Sparassis crispa (Hanabiratake) and its association with beneficial usage.

Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and ß-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential ß-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of ß-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast ß-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.

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September 22, 2019  |  

Assembling the genome of the African wild rice Oryza longistaminata by exploiting synteny in closely related Oryza species.

The African wild rice species Oryza longistaminata has several beneficial traits compared to cultivated rice species, such as resistance to biotic stresses, clonal propagation via rhizomes, and increased biomass production. To facilitate breeding efforts and functional genomics studies, we de-novo assembled a high-quality, haploid-phased genome. Here, we present our assembly, with a total length of 351?Mb, of which 92.2% was anchored onto 12 chromosomes. We detected 34,389 genes and 38.1% of the genome consisted of repetitive content. We validated our assembly by a comparative linkage analysis and by examining well-characterized gene families. This genome assembly will be a useful resource to exploit beneficial alleles found in O. longistaminata. Our results also show that it is possible to generate a high-quality, functionally complete rice genome assembly from moderate SMRT read coverage by exploiting synteny in a closely related Oryza species.

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September 22, 2019  |  

An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila.

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.IMPORTANCEErwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways. Copyright © 2018 Shapiro et al.

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September 22, 2019  |  

Physiological genomics of dietary adaptation in a marine herbivorous fish

Adopting a new diet is a significant evolutionary change and can profoundly affect an animaltextquoterights physiology, biochemistry, ecology, and its genome. To study this evolutionary transition, we investigated the physiology and genomics of digestion of a derived herbivorous fish, the monkeyface prickleback (Cebidichthys violaceus). We sequenced and assembled its genome and digestive transcriptome and revealed the molecular changes related to important dietary enzymes, finding abundant evidence for adaptation at the molecular level. In this species, two gene families experienced expansion in copy number and adaptive amino acid substitutions. These families, amylase, and bile salt activated lipase, are involved digestion of carbohydrates and lipids, respectively. Both show elevated levels of gene expression and increased enzyme activity. Because carbohydrates are abundant in the pricklebacktextquoterights diet and lipids are rare, these findings suggest that such dietary specialization involves both exploiting abundant resources and scavenging rare ones, especially essential nutrients, like essential fatty acids.

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September 22, 2019  |  

How complete are “complete” genome assemblies?-An avian perspective.

The genomics revolution has led to the sequencing of a large variety of nonmodel organisms often referred to as “whole” or “complete” genome assemblies. But how complete are these, really? Here, we use birds as an example for nonmodel vertebrates and find that, although suitable in principle for genomic studies, the current standard of short-read assemblies misses a significant proportion of the expected genome size (7% to 42%; mean 20 ± 9%). In particular, regions with strongly deviating nucleotide composition (e.g., guanine-cytosine-[GC]-rich) and regions highly enriched in repetitive DNA (e.g., transposable elements and satellite DNA) are usually underrepresented in assemblies. However, long-read sequencing technologies successfully characterize many of these underrepresented GC-rich or repeat-rich regions in several bird genomes. For instance, only ~2% of the expected total base pairs are missing in the last chicken reference (galGal5). These assemblies still contain thousands of gaps (i.e., fragmented sequences) because some chromosomal structures (e.g., centromeres) likely contain arrays of repetitive DNA that are too long to bridge with currently available technologies. We discuss how to minimize the number of assembly gaps by combining the latest available technologies with complementary strengths. At last, we emphasize the importance of knowing the location, size and potential content of assembly gaps when making population genetic inferences about adjacent genomic regions.© 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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September 22, 2019  |  

FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan.

A carbapenem-resistant Enterobacter cloacae complex isolated in Tokyo, Japan, produced a carbapenemase that was detected by a Carba NP test and a modified carbapenem inactivation method, but none of the ‘Big Five’ carbapenemase genes was detected by PCR. This study aimed to identify the carbapenemase.Carbapenemase genes were screened by WGS. Next, we generated a recombinant plasmid in which the carbapenemase gene was inserted. We also extracted the carbapenemase gene-carrying plasmid from the E. cloacae complex. The effects of both plasmids on the antibiotic susceptibility of Escherichia coli were then tested. The carbapenemase gene-carrying plasmid in the E. cloacae complex was completely sequenced.A novel carbapenemase gene, blaFRI-4, encoded an amino acid sequence that was 93.2% identical to French imipenemase (FRI-1). E. coli transformed with blaFRI-4 showed reduced carbapenem susceptibility. A complete sequence of the blaFRI-4-carrying 98?508?bp IncFII/IncR plasmid (pTMTA61661) showed that blaFRI-4 and the surrounding region (18.7?kb) were duplicated.The FRI-4-producing E. cloacae complex was isolated in Japan, whereas all other FRI variants have been found in Europe, suggesting that the spread of FRI carbapenemases is global.

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September 22, 2019  |  

Genomic analysis of Picochlorum species reveals how microalgae may adapt to variable environments.

Understanding how microalgae adapt to rapidly changing environments is not only important to science but can help clarify the potential impact of climate change on the biology of primary producers. We sequenced and analyzed the nuclear genome of multiple Picochlorum isolates (Chlorophyta) to elucidate strategies of environmental adaptation. It was previously found that coordinated gene regulation is involved in adaptation to salinity stress, and here we show that gene gain and loss also play key roles in adaptation. We determined the extent of horizontal gene transfer (HGT) from prokaryotes and their role in the origin of novel functions in the Picochlorum clade. HGT is an ongoing and dynamic process in this algal clade with adaptation being driven by transfer, divergence, and loss. One HGT candidate that is differentially expressed under salinity stress is indolepyruvate decarboxylase that is involved in the production of a plant auxin that mediates bacteria-diatom symbiotic interactions. Large differences in levels of heterozygosity were found in diploid haplotypes among Picochlorum isolates. Biallelic divergence was pronounced in P. oklahomensis (salt plains environment) when compared with its closely related sister taxon Picochlorum SENEW3 (brackish water environment), suggesting a role of diverged alleles in response to environmental stress. Our results elucidate how microbial eukaryotes with limited gene inventories expand habitat range from mesophilic to halophilic through allelic diversity, and with minor but important contributions made by HGT. We also explore how the nature and quality of genome data may impact inference of nuclear ploidy.

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September 22, 2019  |  

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown.In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes.Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.

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September 22, 2019  |  

The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans.

Sweet osmanthus (Osmanthus fragrans) is a very popular ornamental tree species throughout Southeast Asia and USA particularly for its extremely fragrant aroma. We constructed a chromosome-level reference genome of O. fragrans to assist in studies of the evolution, genetic diversity, and molecular mechanism of aroma development. A total of over 118?Gb of polished reads was produced from HiSeq (45.1?Gb) and PacBio Sequel (73.35?Gb), giving 100× depth coverage for long reads. The combination of Illumina-short reads, PacBio-long reads, and Hi-C data produced the final chromosome quality genome of O. fragrans with a genome size of 727?Mb and a heterozygosity of 1.45 %. The genome was annotated using de novo and homology comparison and further refined with transcriptome data. The genome of O. fragrans was predicted to have?45,542 genes, of which 95.68 % were functionally annotated. Genome annotation found 49.35 % as the repetitive sequences, with long terminal repeats (LTR) being the richest (28.94 %). Genome evolution analysis indicated the evidence of whole-genome duplication 15 million years ago, which contributed to the current content of 45,242 genes. Metabolic analysis revealed that linalool, a monoterpene is the main aroma compound. Based on the genome and transcriptome, we further demonstrated the direct connection between terpene synthases (TPSs) and the rich aromatic molecules in O. fragrans. We identified three new flower-specific TPS genes, of which the expression coincided with the production of linalool. Our results suggest that the high number of TPS genes and the flower tissue- and stage-specific TPS genes expressions might drive the strong unique aroma production of O. fragrans.

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