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April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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April 21, 2020  |  

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

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April 21, 2020  |  

Whole genomes and transcriptomes reveal adaptation and domestication of pistachio.

Pistachio (Pistacia vera), one of the most important commercial nut crops worldwide, is highly adaptable to abiotic stresses and is tolerant to drought and salt stresses.Here, we provide a draft de novo genome of pistachio as well as large-scale genome resequencing. Comparative genomic analyses reveal stress adaptation of pistachio is likely attributable to the expanded cytochrome P450 and chitinase gene families. Particularly, a comparative transcriptomic analysis shows that the jasmonic acid (JA) biosynthetic pathway plays an important role in salt tolerance in pistachio. Moreover, we resequence 93 cultivars and 14 wild P. vera genomes and 35 closely related wild Pistacia genomes, to provide insights into population structure, genetic diversity, and domestication. We find that frequent genetic admixture occurred among the different wild Pistacia species. Comparative population genomic analyses reveal that pistachio was domesticated about 8000?years ago and suggest that key genes for domestication related to tree and seed size experienced artificial selection.Our study provides insight into genetic underpinning of local adaptation and domestication of pistachio. The Pistacia genome sequences should facilitate future studies to understand the genetic basis of agronomically and environmentally related traits of desert crops.

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April 21, 2020  |  

In the name of the rose: a roadmap for rose research in the genome era.

The recent completion of the rose genome sequence is not the end of a process, but rather a starting point that opens up a whole set of new and exciting activities. Next to a high-quality genome sequence other genomic tools have also become available for rose, including transcriptomics data, a high-density single-nucleotide polymorphism array and software to perform linkage and quantitative trait locus mapping in polyploids. Rose cultivars are highly heterogeneous and diverse. This vast diversity in cultivated roses can be explained through the genetic potential of the genus, introgressions from wild species into commercial tetraploid germplasm and the inimitable efforts of historical breeders. We can now investigate how this diversity can best be exploited and refined in future breeding work, given the rich molecular toolbox now available to the rose breeding community. This paper presents possible lines of research now that rose has entered the genomics era, and attempts to partially answer the question that arises after the completion of any draft genome sequence: ‘Now that we have “the” genome, what’s next?’. Having access to a genome sequence will allow both (fundamental) scientific and (applied) breeding-orientated questions to be addressed. We outline possible approaches for a number of these questions.

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April 21, 2020  |  

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae.The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding.With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus’s strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.

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April 21, 2020  |  

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

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April 21, 2020  |  

Progression of the canonical reference malaria parasite genome from 2002-2019.

Here we describe the ways in which the sequence and annotation of the Plasmodium falciparum reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the P. falciparum research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpretating intra-species sequence diversity. We therefore describe in this publication a population reference genome of P. falciparum, called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. P. falciparum 3D7 will be continued to be maintained with ongoing curation ensuring continual improvements in annotation quality.

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April 21, 2020  |  

Prunus genetics and applications after de novo genome sequencing: achievements and prospects.

Prior to the availability of whole-genome sequences, our understanding of the structural and functional aspects of Prunus tree genomes was limited mostly to molecular genetic mapping of important traits and development of EST resources. With public release of the peach genome and others that followed, significant advances in our knowledge of Prunus genomes and the genetic underpinnings of important traits ensued. In this review, we highlight key achievements in Prunus genetics and breeding driven by the availability of these whole-genome sequences. Within the structural and evolutionary contexts, we summarize: (1) the current status of Prunus whole-genome sequences; (2) preliminary and ongoing work on the sequence structure and diversity of the genomes; (3) the analyses of Prunus genome evolution driven by natural and man-made selection; and (4) provide insight into haploblocking genomes as a means to define genome-scale patterns of evolution that can be leveraged for trait selection in pedigree-based Prunus tree breeding programs worldwide. Functionally, we summarize recent and ongoing work that leverages whole-genome sequences to identify and characterize genes controlling 22 agronomically important Prunus traits. These include phenology, fruit quality, allergens, disease resistance, tree architecture, and self-incompatibility. Translationally, we explore the application of sequence-based marker-assisted breeding technologies and other sequence-guided biotechnological approaches for Prunus crop improvement. Finally, we present the current status of publically available Prunus genomics and genetics data housed mainly in the Genome Database for Rosaceae (GDR) and its updated functionalities for future bioinformatics-based Prunus genetics and genomics inquiry.

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April 21, 2020  |  

Tandem-genotypes: robust detection of tandem repeat expansions from long DNA reads.

Tandemly repeated DNA is highly mutable and causes at least 31 diseases, but it is hard to detect pathogenic repeat expansions genome-wide. Here, we report robust detection of human repeat expansions from careful alignments of long but error-prone (PacBio and nanopore) reads to a reference genome. Our method is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we prioritize pathogenic expansions within the top 10 out of 700,000 tandem repeats in whole genome sequencing data. This may help to elucidate the many genetic diseases whose causes remain unknown.

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April 21, 2020  |  

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.

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April 21, 2020  |  

The complexity of the Fragaria x ananassa (octoploid) transcriptome by single-molecule long-read sequencing.

Strawberry (Fragaria x ananassa) is an allopolyploid species with diverse and complex transcripts. The regulatory mechanisms of fruit development and maturation have been extensively studied; however, little is known about the signaling mechanisms that direct this process in octoploid strawberry (Fragaria x ananassa). Here, we used long-read sequencing (LRS) technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry. We obtained a reference transcriptome with 119,897 unique full-length isoforms, including 2017 new isoforms and 2510 long noncoding RNAs. Based on the genome of the plausible progenitor (Fragaria vesca), 20,229 alternative splicing (AS) events were identified. Using this transcriptome, we found 17,485 differentially expressed transcripts during strawberry fruit development, including 527 transcription factors (TFs) belonging to 41 families. The expression profiles of all members of the auxin, ABA pathway, and anthocyanin biosynthesis gene families were also examined, and many of them were highly expressed at the ripe fruit stage, strongly indicating that the role of those genes is in the regulation of fruit ripening. We produce a high-quality reference transcriptome for octoploid strawberry, including much of the full-length transcript diversity, to help understand the regulatory mechanisms of fruit development and maturation of polyploid species, particularly via elucidation of the biochemical pathways involved in auxin, ABA, and anthocyanin biosynthesis.

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April 21, 2020  |  

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241?bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2?)-Ia, two Tn402-like with ant(3?)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

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April 21, 2020  |  

Genomic characterisation of the new Dickeya fangzhongdai species regrouping plant pathogens and environmental isolates.

The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity.The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100-150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways.This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn’t find any relationship between gene content and phenotypic differences or sharing of environmental habitats.

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April 21, 2020  |  

Comparative genomic and phylogenetic analyses of Populus section Leuce using complete chloroplast genome sequences

Species of Populus section Leuce are distributed throughout most parts of the Northern Hemisphere and have important economic and ecological significance. However, due to frequent hybridization within Leuce, the phylogenetic relationship between species has not been clarified. The chloroplast (cp) genome is characterized by maternal inheritance and relatively conservative mutation rates; thus, it is a powerful tool for building phylogenetic trees. In this study, we used the PacBio SEQUEL software to determine that the cp genome of Populus tomentosa has a length of 156,558 bp including a long single-copy region (84,717 bp), a small single-copy region (16,555 bp), and a pair of inverted repeat regions (27,643 bp). The cp genome contains 131 unique genes, including 37 transfer RNAs, 8 ribosomal RNAs, and 86 protein-coding genes. We compared the cp genomes of seven species of section Leuce and identified five cp DNA markers with >?1% variable sites. Phylogenetic analyses revealed two evolutionary branches for section Leuce. The species with the closest relationship with P. tomenstosa was P. adenopoda, followed by P. alba. These cp genome data will help to determine the cp evolution of section Leuce and further elucidate the origin of P. tomentosa.

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April 21, 2020  |  

Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.

Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene.Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling.Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

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