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September 22, 2019  |  

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.© 2017 BSPP AND JOHN WILEY & SONS LTD.

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September 22, 2019  |  

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Genome biology of a novel lineage of planctomycetes widespread in anoxic aquatic environments.

Anaerobic strains affiliated with a novel order-level lineage of the Phycisphaerae class were retrieved from the suboxic zone of a hypersaline cyanobacterial mat and anoxic sediments of solar salterns. Genome sequences of five isolates were obtained and compared with metagenome-assembled genomes representing related uncultured bacteria from various anoxic aquatic environments. Gene content surveys suggest a strictly fermentative saccharolytic metabolism for members of this lineage, which could be confirmed by the phenotypic characterization of isolates. Genetic analyses indicate that the retrieved isolates do not have a canonical origin of DNA replication, but initiate chromosome replication at alternative sites possibly leading to an accelerated evolution. Further potential factors driving evolution and speciation within this clade include genome reduction by metabolic specialization and rearrangements of the genome by mobile genetic elements, which have a high prevalence in strains from hypersaline sediments and mats. Based on genetic and phenotypic data a distinct group of strictly anaerobic heterotrophic planctomycetes within the Phycisphaerae class could be assigned to a novel order that is represented by the proposed genus Sedimentisphaera gen. nov. comprising two novel species, S. salicampi gen. nov., sp. nov. and S. cyanobacteriorum gen. nov., sp. nov.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019  |  

npInv: accurate detection and genotyping of inversions using long read sub-alignment.

Detection of genomic inversions remains challenging. Many existing methods primarily target inzversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored.We present npInv, a novel tool specifically for detecting and genotyping NAHR inversion using long read sub-alignment of long read sequencing data. We benchmark npInv with other tools in both simulation and real data. We use npInv to generate a whole-genome inversion map for NA12878 consisting of 30 NAHR inversions (of which 15 are novel), including all previously known NAHR mediated inversions in NA12878 with flanking IR less than 7kb. Our genotyping accuracy on this dataset was 94%. We used PCR to confirm the presence of two of these novel inversions. We show that there is a near linear relationship between the length of flanking IR and the minimum inversion size, without inverted repeats.The application of npInv shows high accuracy in both simulation and real data. The results give deeper insight into understanding inversion.

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September 22, 2019  |  

The hpRNA/RNAi pathway is essential to resolve intragenomic conflict in the Drosophila male germline.

Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Genomic insights into host adaptation between the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei).

Plant fungal pathogens can rapidly evolve and adapt to new environmental conditions in response to sudden changes of host populations in agro-ecosystems. However, the genomic basis of their host adaptation, especially at the forma specialis level, remains unclear.We sequenced two isolates each representing Puccinia striiformis f. sp. tritici (Pst) and P. striiformis f. sp. hordei (Psh), different formae speciales of the stripe rust fungus P. striiformis highly adapted to wheat and barley, respectively. The divergence of Pst and Psh, estimated to start 8.12 million years ago, has been driven by high nucleotide mutation rates. The high genomic variation within dikaryotic urediniospores of P. striiformis has provided raw genetic materials for genome evolution. No specific gene families have enriched in either isolate, but extensive gene loss events have occurred in both Pst and Psh after the divergence from their most recent common ancestor. A large number of isolate-specific genes were identified, with unique genomic features compared to the conserved genes, including 1) significantly shorter in length; 2) significantly less expressed; 3) significantly closer to transposable elements; and 4) redundant in pathways. The presence of specific genes in one isolate (or forma specialis) was resulted from the loss of the homologues in the other isolate (or forma specialis) by the replacements of transposable elements or losses of genomic fragments. In addition, different patterns and numbers of telomeric repeats were observed between the isolates.Host adaptation of P. striiformis at the forma specialis level is a complex pathogenic trait, involving not only virulence-related genes but also other genes. Gene loss, which might be adaptive and driven by transposable element activities, provides genomic basis for host adaptation of different formae speciales of P. striiformis.

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September 22, 2019  |  

Ring synthetic chromosome V SCRaMbLE.

Structural variations (SVs) exert important functional impacts on biological phenotypic diversity. Here we show a ring synthetic yeast chromosome V (ring_synV) can be used to continuously generate complex genomic variations and improve the production of prodeoxyviolacein (PDV) by applying Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) in haploid yeast cells. The SCRaMbLE of ring_synV generates aneuploid yeast strains with increased PDV productivity, and we identify aneuploid chromosome I, III, VI, XII, XIII, and ring_synV. The neochromosome of SCRaMbLEd ring_synV generated more unbalanced forms of variations, including duplication, insertions, and balanced forms of translocations and inversions than its linear form. Furthermore, of the 29 novel SVs detected, 11 prompted the PDV biosynthesis; and the deletion of uncharacterized gene YER182W is related to the improvement of the PDV. Overall, the SCRaMbLEing ring_synV embraces the evolution of the genome by modifying the chromosome number, structure, and organization, identifying targets for phenotypic comprehension.

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September 22, 2019  |  

Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles.

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis’ extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.

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September 22, 2019  |  

Ma orthologous genes in Prunus spp. shed light on a noteworthy NBS-LRR cluster conferring differential resistance to root-knot nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

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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  |  

Genomic assemblies of newly sequenced Trypanosoma cruzi strains reveal new genomic expansion and greater complexity.

Chagas disease is a complex illness caused by the protozoan Trypanosoma cruzi displaying highly diverse clinical outcomes. In this sense, the genome sequence elucidation and comparison between strains may lead to disease understanding. Here, two new T. cruzi strains, have been sequenced, Y using Illumina and Bug2148 using PacBio, assembled, analyzed and compared with the T. cruzi annotated genomes available to date. The assembly stats from the new sequences show effective improvement of T. cruzi genome over the actual ones. Such as, the largest contig assembled (1.3?Mb in Bug2148) in de novo attempts and the highest mean assembly coverage (71X for Y). Our analysis reveals a new genomic expansion and greater complexity for those multi-copy gene families related to infection process and disease development, such as Trans-sialidases, Mucins and Mucin Associated Surface Proteins, among others. On one side, we demonstrate that multi-copy gene families are located near telomeric regions of the “chromosome-like” 1.3?Mb contig assembled of Bug2148, where they likely suffer high evolutive pressure. On the other hand, we identified several strain-specific single copy genes that might help to understand the differences in infectivity and physiology among strains. In summary, our results indicate that T. cruzi has a complex genomic architecture that may have promoted its evolution.

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September 22, 2019  |  

Multisite de novo mutations in human offspring after paternal exposure to ionizing radiation.

A genome-wide evaluation of the effects of ionizing radiation on mutation induction in the mouse germline has identified multisite de novo mutations (MSDNs) as marker for previous exposure. Here we present the results of a small pilot study of whole genome sequencing in offspring of soldiers who served in radar units on weapon systems that were emitting high-frequency radiation. We found cases of exceptionally high MSDN rates as well as an increased mean in our cohort: While a MSDN mutation is detected in average in 1 out of 5 offspring of unexposed controls, we observed 12 MSDNs in altogether 18 offspring, including a family with 6 MSDNs in 3 offspring. Moreover, we found two translocations, also resulting from neighboring mutations. Our findings indicate that MSDNs might be suited in principle for the assessment of DNA damage from ionizing radiation also in humans. However, as exact person-related dose values in risk groups are usually not available, the interpretation of MSDNs in single families would benefit from larger molecular epidemiologic studies on this new biomarker.

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September 22, 2019  |  

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

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September 22, 2019  |  

Bacillus wiedmannii biovar thuringiensis: A specialized mosquitocidal pathogen with plasmids from diverse origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous e?orts have been undertaken to allow presumptive di?erentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490?kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.

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September 22, 2019  |  

The genomic basis of color pattern polymorphism in the Harlequin ladybird.

Many animal species comprise discrete phenotypic forms. A common example in natural populations of insects is the occurrence of different color patterns, which has motivated a rich body of ecological and genetic research [1-6]. The occurrence of dark, i.e., melanic, forms displaying discrete color patterns is found across multiple taxa, but the underlying genomic basis remains poorly characterized. In numerous ladybird species (Coccinellidae), the spatial arrangement of black and red patches on adult elytra varies wildly within species, forming strikingly different complex color patterns [7, 8]. In the harlequin ladybird, Harmonia axyridis, more than 200 distinct color forms have been described, which classic genetic studies suggest result from allelic variation at a single, unknown, locus [9, 10]. Here, we combined whole-genome sequencing, population-based genome-wide association studies, gene expression, and functional analyses to establish that the transcription factor Pannier controls melanic pattern polymorphism in H. axyridis. We show that pannier is necessary for the formation of melanic elements on the elytra. Allelic variation in pannier leads to protein expression in distinct domains on the elytra and thus determines the distinct color patterns in H. axyridis. Recombination between pannier alleles may be reduced by a highly divergent sequence of ~170 kb in the cis-regulatory regions of pannier, with a 50 kb inversion between color forms. This most likely helps maintain the distinct alleles found in natural populations. Thus, we propose that highly variable discrete color forms can arise in natural populations through cis-regulatory allelic variation of a single gene. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

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