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September 22, 2019

Deciphering lignocellulose deconstruction by the white rot fungus Irpex lacteus based on genomic and transcriptomic analyses.

Irpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.Irpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.The selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

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September 22, 2019

Targeted long-read sequencing of a locus under long-term balancing selection in Capsella.

Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10-5 A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach. Copyright © 2018 Bachmann et al.

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September 22, 2019

In vitro culture of the insect endosymbiont Spiroplasma poulsonii highlights bacterial genes involved in host-symbiont interaction.

Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with several Drosophila species. S. poulsonii strongly affects its host’s physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic Spiroplasma in vitro have failed so far. Here, we developed a method to sustain the in vitro culture of S. poulsonii by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic Spiroplasma species. Last, by comparing the transcriptome of the in vitro culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic Spiroplasma and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners.IMPORTANCE The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. Spiroplasma poulsonii is an endosymbiont of Drosophila that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting in vitro culture of Spiroplasma and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions. Copyright © 2018 Masson et al.

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September 22, 2019

Whole genome sequencing of greater amberjack (Seriola dumerili) for SNP identification on aligned scaffolds and genome structural variation analysis using parallel resequencing

Greater amberjack (Seriola dumerili) is distributed in tropical and temperate waters worldwide and is an important aquaculture fish. We carried out de novo sequencing of the greater amberjack genome to construct a reference genome sequence to identify single nucleotide polymorphisms (SNPs) for breeding amberjack by marker-assisted or gene-assisted selection as well as to identify functional genes for biological traits. We obtained 200 times coverage and constructed a high-quality genome assembly using next generation sequencing technology. The assembled sequences were aligned onto a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map by sequence homology. A total of 215 of the longest amberjack sequences, with a total length of 622.8?Mbp (92% of the total length of the genome scaffolds), were lined up on the yellowtail RH map. We resequenced the whole genomes of 20 greater amberjacks and mapped the resulting sequences onto the reference genome sequence. About 186,000 nonredundant SNPs were successfully ordered on the reference genome. Further, we found differences in the genome structural variations between two greater amberjack populations using BreakDancer. We also analyzed the greater amberjack transcriptome and mapped the annotated sequences onto the reference genome sequence.

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September 22, 2019

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol.

The demand for cellulosic biofuels is on the rise because of the anticipation for sustainable energy and less greenhouse gas emissions in the future. However, production of cellulosic biofuels, especially cellulosic butanol, has been hampered by the lack of potent microbes that are capable of converting cellulosic biomass into biofuels. We report a wild-type Thermoanaerobacterium thermosaccharolyticum strain TG57, which is capable of using microcrystalline cellulose directly to produce butanol (1.93 g/liter) as the only final product (without any acetone or ethanol produced), comparable to that of engineered microbes thus far. Strain TG57 exhibits significant advances including unique genes responsible for a new butyrate synthesis pathway, no carbon catabolite repression, and the absence of genes responsible for acetone synthesis (which is observed as the main by-product in most Clostridium strains known today). Furthermore, the use of glucose analog 2-deoxyglucose posed a selection pressure to facilitate isolation of strain TG57 with deletion/silencing of carbon catabolite repressor genes-the ccr and xylR genes-and thus is able to simultaneously ferment glucose, xylose, and arabinose to produce butanol (7.33 g/liter) as the sole solvent. Combined analysis of genomic and transcriptomic data revealed unusual aspects of genome organization, numerous determinants for unique bioconversions, regulation of central metabolic pathways, and distinct transcriptomic profiles. This study provides a genome-level understanding of how cellulose is metabolized by T. thermosaccharolyticum and sheds light on the potential of competitive and sustainable biofuel production.

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September 22, 2019

Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria.

The chemical diversity of physiological DNA modifications has expanded with the identification of phosphorothioate (PT) modification in which the nonbridging oxygen in the sugar-phosphate backbone of DNA is replaced by sulfur. Together with DndFGH as cognate restriction enzymes, DNA PT modification, which is catalyzed by the DndABCDE proteins, functions as a bacterial restriction-modification (R-M) system that protects cells against invading foreign DNA. However, the occurrence of dnd systems across a large number of bacterial genomes and their functions other than R-M are poorly understood. Here, a genomic survey revealed the prevalence of bacterial dnd systems: 1,349 bacterial dnd systems were observed to occur sporadically across diverse phylogenetic groups, and nearly half of these occur in the form of a solitary dndBCDE gene cluster that lacks the dndFGH restriction counterparts. A phylogenetic analysis of 734 complete PT R-M pairs revealed the coevolution of M and R components, despite the observation that several PT R-M pairs appeared to be assembled from M and R parts acquired from distantly related organisms. Concurrent epigenomic analysis, transcriptome analysis, and metabolome characterization showed that a solitary PT modification contributed to the overall cellular redox state, the loss of which perturbed the cellular redox balance and induced Pseudomonas fluorescens to reconfigure its metabolism to fend off oxidative stress. An in vitro transcriptional assay revealed altered transcriptional efficiency in the presence of PT DNA modification, implicating its function in epigenetic regulation. These data suggest the versatility of PT in addition to its involvement in R-M protection.

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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

The genome sequence of a new strain of Mycobacterium ulcerans ecovar Liflandii, emerging as a sturgeon pathogen

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) is emerging as a non-mycobacterial pathogen in amphibians. Here, we make the first report on the prevalence of a new strain of MuLiflandii infection in Chinese sturgeon. All the diseased fish showed the classic clinical symptoms of ascites and/or muscle ulceration. A new slow-growing and acid-fast bacillus ASM001 strain was obtained from the ascites of infected fish; this strain demonstrated pathogenicity when tested in hybrid sturgeon. The complete genome sequence of MuLiflandii ASM001 is a circular chromosome of 6,167,296?bp, with a G?+?C content of 65.57%, containing 4518 predicted coding DNA sequences and 999 pseudo-genes, 3 rRNA operons, and 47 transfer RNA sequences. In addition, we found 245 copies of IS2404, 34 microsatellites, and 36 CRISPR sequences in the whole MuLiflandii ASM001 genome. Among the predicted genes of MuLiflandii ASM001, we found orthologs of 203 virulence factors of clinical MuLiflandii 128FXT operating in host cell invasion, modulation of phagocyte function, and survival inside the macrophages. These virulence factor candidates provide a key basis for understanding their pathogenic mechanisms at the molecular level. A comparative analysis that used complete, existing genomes showed that MuLiflandii ASM001 has high synteny with MuLiflandii 128FXT. We anticipate the availability of the complete MuLiflandii ASM001 genome sequence will provide a valuable resource for comparative genomic studies of MuLiflandii isolates, as well as provide new insights into the host, ecological, and functional diversity of the genus Mycobacterium.

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September 22, 2019

Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

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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

Genomic diversity of Taylorella equigenitalis introduced into the United States from 1978 to 2012.

Contagious equine metritis is a disease of worldwide concern in equids. The United States is considered to be free of the disease although sporadic outbreaks have occurred over the last few decades that were thought to be associated with the importation of horses. The objective of this study was to create finished, reference quality genomes that characterize the diversity of Taylorella equigenitalis isolates introduced into the USA, and identify their differences. Five isolates of T. equigenitalis associated with introductions into the USA from unique sources were sequenced using both short and long read chemistries allowing for complete assembly and annotation. These sequences were compared to previously published genomes as well as the short read sequences of the 200 isolates in the National Veterinary Services Laboratories’ diagnostic repository to identify unique regions and genes, potential virulence factors, and characterize diversity. The 5 genomes varied in size by up to 100,000 base pairs, but averaged 1.68 megabases. The majority of that diversity in size can be explained by repeat regions and 4 main regions of difference, which ranged in size from 15,000 to 45,000 base pairs. The first region of difference contained mostly hypothetical proteins, the second contained the CRISPR, the third contained primarily hemagglutinin proteins, and the fourth contained primarily segments of a type IV secretion system. As expected and previously reported, little evidence of recombination was found within these genomes. Several additional areas of interest were also observed including a mechanism for streptomycin resistance and other virulence factors. A SNP distance comparison of the T. equigenitalis isolates and Mycobacterium tuberculosis complex (MTBC) showed that relatively, T. equigenitalis was a more diverse species than the entirety of MTBC.

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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

Comparative genomic insights into endofungal lifestyles of two bacterial endosymbionts, Mycoavidus cysteinexigens and Burkholderia rhizoxinica.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.

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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

Microsatellite polymorphism in the endangered snail kite reveals a panmictic, low diversity population

Genetic structure and genetic diversity are key population characteristics that can inform conservation decisions, such as delineating management units or assessing potential risks for inbreeding depression. Evidence of genetic structuring or low genetic diversity in the critically endangered snail kite (Rostrhamus sociabilis plumbeus) would have implications for monitoring and planning decisions. Recent work on understanding connectivity across the snail kite range indicated that there is less dispersal between northern and southern parts of the current range, and that dispersal is shaped by individual habitat preference. We examine whether there is neutral genetic structure and the amount of genetic variation in the population by non-lethally sampling 235 nestlings from unique nests across the entire breeding range between 2013 and 2014. Data on 15 microsatellite revealed low diversity (e.g., Na?=?2.54, He?=?0.37) and range-wide panmixia based on AMOVA, Bayesian clustering, spatial autocorrelation, isolation by distance, and spatially explicit ordination analyses. Our results emphasize that long-term recovery goals and management strategies should be based on viewing snail kites as a single genetic population, despite evidence for non-random dispersal between wetlands over ecological time scales. These results also highlight the need to understand potential effects of low genetic diversity on population dynamics and viability of snail kites. More broadly, these results add to the growing evidence for potential discrepancies between dispersal and genetic patterns, emphasizing that care should be taken if using one to interpret the other, particularly for widely-ranging species.

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