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September 22, 2019

Long-read genome sequence and assembly of Leptopilina boulardi: a specialist Drosophila parasitoid

Background: Leptopilina boulardi is a specialist parasitoid belonging to the order Hymenoptera, which attacks the larval stages of Drosophila. The Leptopilina genus has enormous value in the biological control of pests as well as in understanding several aspects of host-parasitoid biology. However, none of the members of Figitidae family has their genomes sequenced. In order to improve the understanding of the parasitoid wasps by generating genomic resources, we sequenced the whole genome of L. boulardi. Findings: Here, we report a high quality genome of L. boulardi, assembled from 70Gb of Illumina reads and 10.5Gb of PacBio reads, forming a total coverage of 230X. The 375Mb draft genome has an N50 of 275Kb with 6315 scaffolds >500bp, and encompasses >95% complete BUSCOs. The GC% of the genome is 28.26%, and RepeatMasker identified 868105 repeat elements covering 43.9% of the assembly. A total of 25259 protein-coding genes were predicted using a combination of ab-initio and RNA-Seq based methods, with an average gene size of 3.9Kb. 78.11% of the predicted genes could be annotated with at least one function. Conclusion: Our study provides a highly reliable assembly of this parasitoid wasp, which will be a valuable resource to researchers studying parasitoids. In particular, it can help delineate the host-parasitoid mechanisms that are part of the Drosophila-Leptopilina model system.

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September 22, 2019

Stress-adaptive responses associated with high-level carbapenem resistance in KPC-producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) organisms have emerged to become a major global public health threat among antimicrobial resistant bacterial human pathogens. Little is known about how CREs emerge. One characteristic phenotype of CREs is heteroresistance, which is clinically associated with treatment failure in patients given a carbapenem. Through in vitro whole-transcriptome analysis we tracked gene expression over time in two different strains (BR7, BR21) of heteroresistant KPC-producing Klebsiella pneumoniae, first exposed to a bactericidal concentration of imipenem followed by growth in drug-free medium. In both strains, the immediate response was dominated by a shift in expression of genes involved in glycolysis toward those involved in catabolic pathways. This response was followed by global dampening of transcriptional changes involving protein translation, folding and transport, and decreased expression of genes encoding critical junctures of lipopolysaccharide biosynthesis. The emerged high-level carbapenem-resistant BR21 subpopulation had a prophage (IS1) disrupting ompK36 associated with irreversible OmpK36 porin loss. On the other hand, OmpK36 loss in BR7 was reversible. The acquisition of high-level carbapenem resistance by the two heteroresistant strains was associated with distinct and shared stepwise transcriptional programs. Carbapenem heteroresistance may emerge from the most adaptive subpopulation among a population of cells undergoing a complex set of stress-adaptive responses.

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September 22, 2019

Dynamic evolution of a-gliadin prolamin gene family in homeologous genomes of hexaploid wheat.

Wheat Gli-2 loci encode complex groups of a-gliadin prolamins that are important for breadmaking, but also major triggers of celiac disease (CD). Elucidation of a-gliadin evolution provides knowledge to produce wheat with better end-use properties and reduced immunogenic potential. The Gli-2 loci contain a large number of tandemly duplicated genes and highly repetitive DNA, making sequence assembly of their genomic regions challenging. Here, we constructed high-quality sequences spanning the three wheat homeologous a-gliadin loci by aligning PacBio-based sequence contigs with BioNano genome maps. A total of 47 a-gliadin genes were identified with only 26 encoding intact full-length protein products. Analyses of a-gliadin loci and phylogenetic tree reconstruction indicate significant duplications of a-gliadin genes in the last ~2.5 million years after the divergence of the A, B and D genomes, supporting its rapid lineage-independent expansion in different Triticeae genomes. We showed that dramatic divergence in expression of a-gliadin genes could not be attributed to sequence variations in the promoter regions. The study also provided insights into the evolution of CD epitopes and identified a single indel event in the hexaploid wheat D genome that likely resulted in the generation of the highly toxic 33-mer CD epitope.

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September 22, 2019

Targeted sequencing by gene synteny, a new strategy for polyploid species: sequencing and physical structure of a complex sugarcane region.

Sugarcane exhibits a complex genome mainly due to its aneuploid nature and high ploidy level, and sequencing of its genome poses a great challenge. Closely related species with well-assembled and annotated genomes can be used to help assemble complex genomes. Here, a stable quantitative trait locus (QTL) related to sugar accumulation in sorghum was successfully transferred to the sugarcane genome. Gene sequences related to this QTL were identified in silico from sugarcane transcriptome data, and molecular markers based on these sequences were developed to select bacterial artificial chromosome (BAC) clones from the sugarcane variety SP80-3280. Sixty-eight BAC clones containing at least two gene sequences associated with the sorghum QTL were sequenced using Pacific Biosciences (PacBio) technology. Twenty BAC sequences were found to be related to the syntenic region, of which nine were sufficient to represent this region. The strategy we propose is called “targeted sequencing by gene synteny,” which is a simpler approach to understanding the genome structure of complex genomic regions associated with traits of interest.

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September 22, 2019

Cross-species comparison of the gut: Differential gene expression sheds light on biological differences in closely related tenebrionids.

The gut is one of the primary interfaces between an insect and its environment. Understanding gene expression profiles in the insect gut can provide insight into interactions with the environment as well as identify potential control methods for pests. We compared the expression profiles of transcripts from the gut of larval stages of two coleopteran insects, Tenebrio molitor and Tribolium castaneum. These tenebrionids have different life cycles, varying in the duration and number of larval instars. T. castaneum has a sequenced genome and has been a model for coleopterans, and we recently obtained a draft genome for T. molitor. We assembled gut transcriptome reads from each insect to their respective genomes and filtered mapped reads to RPKM>1, yielding 11,521 and 17,871 genes in the T. castaneum and T. molitor datasets, respectively. There were identical GO terms in each dataset, and enrichment analyses also identified shared GO terms. From these datasets, we compiled an ortholog list of 6907 genes; 45% of the total assembled reads from T. castaneum were found in the top 25 orthologs, but only 27% of assembled reads were found in the top 25 T. molitor orthologs. There were 2281 genes unique to T. castaneum, and 2088 predicted genes unique to T. molitor, although improvements to the T. molitor genome will likely reduce these numbers as more orthologs are identified. We highlight a few unique genes in T. castaneum or T. molitor that may relate to distinct biological functions. A large number of putative genes expressed in the larval gut with uncharacterized functions (36 and 68% from T. castaneum and T. molitor, respectively) support the need for further research. These data are the first step in building a comprehensive understanding of the physiology of the gut in tenebrionid insects, illustrating commonalities and differences that may be related to speciation and environmental adaptation. Published by Elsevier Ltd.

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September 22, 2019

Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.

Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12. Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2, indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion.

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September 22, 2019

Benefit from decline: the primary transcriptome of Alteromonas macleodii str. Te101 during Trichodesmium demise.

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63?Mb and a single plasmid of 237?kb. Increasing salinities to =43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101_05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment.

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September 22, 2019

Autologous cell therapy approach for Duchenne muscular dystrophy using PiggyBac transposons and mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Genome sequence, assembly and characterization of two Metschnikowia fructicola strains used as biocontrol agents of postharvest diseases.

The yeast Metschnikowia fructicola was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product “Shemer.” Several mechanisms of action by which M. fructicola inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of M. fructicola using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of M. fructicola proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two M. fructicola strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among M. fructicola genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.

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September 22, 2019

Comparative genomics of the wheat fungal pathogen Pyrenophora tritici-repentis reveals chromosomal variations and genome plasticity.

Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen.Here, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets.Whole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.

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September 22, 2019

Ginseng Genome Database: an open-access platform for genomics of Panax ginseng.

The ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous plant that has been used in traditional oriental medicine for thousands of years. Ginsenosides, which have significant pharmacological effects on human health, are the foremost bioactive constituents in this plant. Having realized the importance of this plant to humans, an integrated omics resource becomes indispensable to facilitate genomic research, molecular breeding and pharmacological study of this herb.The first draft genome sequences of P. ginseng cultivar “Chunpoong” were reported recently. Here, using the draft genome, transcriptome, and functional annotation datasets of P. ginseng, we have constructed the Ginseng Genome Database http://ginsengdb.snu.ac.kr /, the first open-access platform to provide comprehensive genomic resources of P. ginseng. The current version of this database provides the most up-to-date draft genome sequence (of approximately 3000 Mbp of scaffold sequences) along with the structural and functional annotations for 59,352 genes and digital expression of genes based on transcriptome data from different tissues, growth stages and treatments. In addition, tools for visualization and the genomic data from various analyses are provided. All data in the database were manually curated and integrated within a user-friendly query page.This database provides valuable resources for a range of research fields related to P. ginseng and other species belonging to the Apiales order as well as for plant research communities in general. Ginseng genome database can be accessed at http://ginsengdb.snu.ac.kr /.

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September 22, 2019

Draft genome of the Peruvian scallop Argopecten purpuratus.

The Peruvian scallop, Argopecten purpuratus, is mainly cultured in southern Chile and Peru was introduced into China in the last century. Unlike other Argopecten scallops, the Peruvian scallop normally has a long life span of up to 7 to 10 years. Therefore, researchers have been using it to develop hybrid vigor. Here, we performed whole genome sequencing, assembly, and gene annotation of the Peruvian scallop, with an important aim to develop genomic resources for genetic breeding in scallops.A total of 463.19-Gb raw DNA reads were sequenced. A draft genome assembly of 724.78 Mb was generated (accounting for 81.87% of the estimated genome size of 885.29 Mb), with a contig N50 size of 80.11 kb and a scaffold N50 size of 1.02 Mb. Repeat sequences were calculated to reach 33.74% of the whole genome, and 26,256 protein-coding genes and 3,057 noncoding RNAs were predicted from the assembly.We generated a high-quality draft genome assembly of the Peruvian scallop, which will provide a solid resource for further genetic breeding and for the analysis of the evolutionary history of this economically important scallop.

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September 22, 2019

Evolution of sequence type 4821 clonal complex meningococcal strains in China from prequinolone to quinolone era, 1972-2013.

The expansion of hypervirulent sequence type 4821 clonal complex (CC4821) lineage Neisseria meningitidis bacteria has led to a shift in meningococcal disease epidemiology in China, from serogroup A (MenA) to MenC. Knowledge of the evolution and genetic origin of the emergent MenC strains is limited. In this study, we subjected 76 CC4821 isolates collected across China during 1972-1977 and 2005-2013 to phylogenetic analysis, traditional genotyping, or both. We show that successive recombination events within genes encoding surface antigens and acquisition of quinolone resistance mutations possibly played a role in the emergence of CC4821 as an epidemic clone in China. MenC and MenB CC4821 strains have spread across China and have been detected in several countries in different continents. Capsular switches involving serogroups B and C occurred among epidemic strains, raising concerns regarding possible increases in MenB disease, given that vaccines in use in China do not protect against MenB.

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September 22, 2019

Gene presence-absence polymorphism in castrating anther-smut fungi: Recent gene Gains and Phylogeographic Structure.

Gene presence-absence polymorphisms segregating within species are a significant source of genetic variation but have been little investigated to date in natural populations. In plant pathogens, the gain or loss of genes encoding proteins interacting directly with the host, such as secreted proteins, probably plays an important role in coevolution and local adaptation. We investigated gene presence-absence polymorphism in populations of two closely related species of castrating anther-smut fungi, Microbotryum lychnidis-dioicae (MvSl) and M. silenes-dioicae (MvSd), from across Europe, on the basis of Illumina genome sequencing data and high-quality genome references. We observed presence-absence polymorphism for 186 autosomal genes (2% of all genes) in MvSl, and only 51 autosomal genes in MvSd. Distinct genes displayed presence-absence polymorphism in the two species. Genes displaying presence-absence polymorphism were frequently located in subtelomeric and centromeric regions and close to repetitive elements, and comparison with outgroups indicated that most were present in a single species, being recently acquired through duplications in multiple-gene families. Gene presence-absence polymorphism in MvSl showed a phylogeographic structure corresponding to clusters detected based on SNPs. In addition, gene absence alleles were rare within species and skewed toward low-frequency variants. These findings are consistent with a deleterious or neutral effect for most gene presence-absence polymorphism. Some of the observed gene loss and gain events may however be adaptive, as suggested by the putative functions of the corresponding encoded proteins (e.g., secreted proteins) or their localization within previously identified selective sweeps. The adaptive roles in plant and anther-smut fungi interactions of candidate genes however need to be experimentally tested in future studies.

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September 22, 2019

Comparative genomics of bdelloid rotifers: Insights from desiccating and nondesiccating species.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer.

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