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September 22, 2019

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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September 22, 2019

The draft genome assembly of Dermatophagoides pteronyssinus supports identification of novel allergen isoforms in Dermatophagoides species.

Background: Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) are highly similar disease-asso- ciated mites with frequently overlapping geographic distributions. A draft genome of DP was assembled to identify the candidate allergens in DP that are homologous to those in DF, investigate allergen isoforms, and facilitate comparisons with related Acari. Methods: PacBio and Illumina whole-genome sequencing was performed on DP. Assembly and reconstruction of the genomes were optimized for isoform identification in a heterogeneous population. Bioinformatic analyses of Acari genomes were performed. Results: The predicted size of the DP nuclear genome is 52.5 Mb. A predicted set of 19,368 proteins was identified, including all 19 currently recognized allergens from this species. Orthologs for 12 allergens established for DF were found. The population of DP mites showed a high level of heterozygosity that allowed the identification of 43 new isoforms for both established and candidate allergens in DP including a new isoform for the major allergen Der p 23. Reanalyzing the previous DF data assuming heterozygosity, 14 new allergen isoforms could be identified. Some new isoforms were observed in both species, suggesting that these isoforms predated speciation. The high quality of both genomes allowed an examination of synteny which showed that many allergen orthologs are physically clustered but with species-specific exon/intron structures. Comparative genomic analyses of other Acariformes mites showed that most of the allergen homologs are widely conserved within this Superorder. Conclusions: Candidate allergens in DP were identified to facilitate future serological studies. While DP and DF are highly similar genetically, species-specific allergen isoforms exist to facilitate molecular differentiation.

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September 22, 2019

The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest,Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families revealT. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, andT. nisiRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. TheT. nigenome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.© 2018, Fu et al.

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September 22, 2019

Redkmer: An Assembly-Free Pipeline for the Identification of Abundant and Specific X-Chromosome Target Sequences for X-Shredding by CRISPR Endonucleases.

CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.

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September 22, 2019

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young ‘AA’ subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 ‘Miracle Rice’, which relieved famine and drove the Green Revolution in Asia 50 years ago.

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September 22, 2019

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei.

For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.© The Authors 2017. Published by Oxford University Press.

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September 22, 2019

By land, air, and sea: hemipteran diversity through the genomic lens

Thanks to a recent spate of sequencing projects, the Hemiptera are the first hemimetabolous insect order to achieve a critical mass of species with sequenced genomes, establishing the basis for comparative genomics of the bugs. However, as the most speciose hemimetabolous order, there is still a vast swathe of the hemipteran phylogeny that awaits genomic representation across subterranean, terrestrial, and aquatic habitats, and with lineage-specific and developmentally plastic cases of both wing polyphenisms and flightlessness. In this review, we highlight opportunities for taxonomic sampling beyond obvious pest species candidates, motivated by intriguing biological features of certain groups as well as the rich research tradition of ecological, physiological, developmental, and particularly cytogenetic investigation that spans the diversity of the Hemiptera.

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September 22, 2019

The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys’ unique biology and evolutionary/comparative perspective.

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September 22, 2019

LTR_retriever: A highly accurate and sensitive program for identification of long terminal repeat retrotransposons.

Long terminal repeat retrotransposons (LTR-RTs) are prevalent in plant genomes. The identification of LTR-RTs is critical for achieving high-quality gene annotation. Based on the well-conserved structure, multiple programs were developed for the de novo identification of LTR-RTs; however, these programs are associated with low specificity and high false discovery rates. Here, we report LTR_retriever, a multithreading-empowered Perl program that identifies LTR-RTs and generates high-quality LTR libraries from genomic sequences. LTR_retriever demonstrated significant improvements by achieving high levels of sensitivity (91%), specificity (97%), accuracy (96%), and precision (90%) in rice (Oryza sativa). LTR_retriever is also compatible with long sequencing reads. With 40k self-corrected PacBio reads equivalent to 4.5× genome coverage in Arabidopsis (Arabidopsis thaliana), the constructed LTR library showed excellent sensitivity and specificity. In addition to canonical LTR-RTs with 5′-TG…CA-3′ termini, LTR_retriever also identifies noncanonical LTR-RTs (non-TGCA), which have been largely ignored in genome-wide studies. We identified seven types of noncanonical LTRs from 42 out of 50 plant genomes. The majority of noncanonical LTRs areCopiaelements, with which the LTR is four times shorter than that of otherCopiaelements, which may be a result of their target specificity. Strikingly, non-TGCACopiaelements are often located in genic regions and preferentially insert nearby or within genes, indicating their impact on the evolution of genes and their potential as mutagenesis tools.© 2018 American Society of Plant Biologists. All Rights Reserved.

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September 22, 2019

Bat biology, genomes, and the Bat1K project: To generate chromosome-level genomes for all living bat species.

Bats are unique among mammals, possessing some of the rarest mammalian adaptations, including true self-powered flight, laryngeal echolocation, exceptional longevity, unique immunity, contracted genomes, and vocal learning. They provide key ecosystem services, pollinating tropical plants, dispersing seeds, and controlling insect pest populations, thus driving healthy ecosystems. They account for more than 20% of all living mammalian diversity, and their crown-group evolutionary history dates back to the Eocene. Despite their great numbers and diversity, many species are threatened and endangered. Here we announce Bat1K, an initiative to sequence the genomes of all living bat species (n~1,300) to chromosome-level assembly. The Bat1K genome consortium unites bat biologists (>148 members as of writing), computational scientists, conservation organizations, genome technologists, and any interested individuals committed to a better understanding of the genetic and evolutionary mechanisms that underlie the unique adaptations of bats. Our aim is to catalog the unique genetic diversity present in all living bats to better understand the molecular basis of their unique adaptations; uncover their evolutionary history; link genotype with phenotype; and ultimately better understand, promote, and conserve bats. Here we review the unique adaptations of bats and highlight how chromosome-level genome assemblies can uncover the molecular basis of these traits. We present a novel sequencing and assembly strategy and review the striking societal and scientific benefits that will result from the Bat1K initiative.

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September 22, 2019

Genetic separation of Listeria monocytogenes causing central nervous system infections in animals.

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains.

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September 22, 2019

The DNA methylome of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

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September 22, 2019

Culture-facilitated comparative genomics of the facultative symbiont Hamiltonella defensa.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.

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September 22, 2019

Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2.

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (?AbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102~4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ?AbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ?AbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ?AbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression.

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September 22, 2019

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides.

The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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