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September 22, 2019

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (Camellia sinensis)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.

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September 22, 2019

The antibody loci of the domestic goat (Capra hircus).

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

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September 22, 2019

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

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September 22, 2019

Solar-panel and parasol strategies shape the proteorhodopsin distribution pattern in marine Flavobacteriia.

Proteorhodopsin (PR) is a light-driven proton pump that is found in diverse bacteria and archaea species, and is widespread in marine microbial ecosystems. To date, many studies have suggested the advantage of PR for microorganisms in sunlit environments. The ecophysiological significance of PR is still not fully understood however, including the drivers of PR gene gain, retention, and loss in different marine microbial species. To explore this question we sequenced 21 marine Flavobacteriia genomes of polyphyletic origin, which encompassed both PR-possessing as well as PR-lacking strains. Here, we show that the possession or alternatively the lack of PR genes reflects one of two fundamental adaptive strategies in marine bacteria. Specifically, while PR-possessing bacteria utilize light energy (“solar-panel strategy”), PR-lacking bacteria exclusively possess UV-screening pigment synthesis genes to avoid UV damage and would adapt to microaerobic environment (“parasol strategy”), which also helps explain why PR-possessing bacteria have smaller genomes than those of PR-lacking bacteria. Collectively, our results highlight the different strategies of dealing with light, DNA repair, and oxygen availability that relate to the presence or absence of PR phototrophy.

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September 22, 2019

DNA N6-adenine methylation in Arabidopsis thaliana.

DNA methylation on N6-adenine (6mA) has recently been found to be a potentially epigenetic mark in several unicellular and multicellular eukaryotes. However, its distribution patterns and potential functions in land plants, which are primary producers for most ecosystems, remain largely unknown. Here we report global profiling of 6mA sites at single-nucleotide resolution in the genome of Arabidopsis thaliana at different developmental stages using single-molecule real-time sequencing. 6mA sites are widely distributed across the Arabidopsis genome and enriched over the pericentromeric heterochromatin regions. 6mA occurs more frequently in gene bodies than intergenic regions. Analysis of 6mA methylomes and RNA sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level and the transition from vegetative to reproductive growth in Arabidopsis. Our results uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a hitherto unknown epigenetic mark in land plants. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

The complete replicons of 16 Ensifer meliloti strains offer insights into intra- and inter-replicon gene transfer, transposon-associated loci, and repeat elements.

Ensifer meliloti (formerly Rhizobium meliloti and Sinorhizobium meliloti) is a model bacterium for understanding legume-rhizobial symbioses. The tripartite genome of E. meliloti consists of a chromosome, pSymA and pSymB, and in some instances strain-specific accessory plasmids. The majority of previous sequencing studies have relied on the use of assemblies generated from short read sequencing, which leads to gaps and assembly errors. Here we used PacBio-based, long-read assemblies and were able to assemble, de novo, complete circular replicons. In this study, we sequenced, de novo-assembled and analysed 10 E. meliloti strains. Sequence comparisons were also done with data from six previously published genomes. We identified genome differences between the replicons, including mol% G+C and gene content, nucleotide repeats, and transposon-associated loci. Additionally, genomic rearrangements both within and between replicons were identified, providing insight into evolutionary processes at the structural level. There were few cases of inter-replicon gene transfer of core genes between the main replicons. Accessory plasmids were more similar to pSymA than to either pSymB or the chromosome, with respect to gene content, transposon content and G+C content. In our population, the accessory plasmids appeared to share an open genome with pSymA, which contains many nodulation- and nitrogen fixation-related genes. This may explain previous observations that horizontal gene transfer has a greater effect on the content of pSymA than pSymB, or the chromosome, and why some rhizobia show unstable nodulation phenotypes on legume hosts.

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September 22, 2019

The Egyptian rousette genome reveals unexpected features of bat antiviral immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Spread of plasmid-encoded NDM-1 and GES-5 carbapenemases among extensively drug-resistant and pandrug-resistant clinical Enterobacteriaceae in Durban, South Africa.

Whole-genome sequence analyses revealed the presence of blaNDM-1 (n = 31), blaGES-5 (n = 8), blaOXA-232 (n = 1), or blaNDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], blaNDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the blaNDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this blaNDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried blaGES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected blaOXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and blaNDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

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September 22, 2019

Integrated proteomics, genomics, metabolomics approaches reveal oxalic acid as pathogenicity factor in Tilletia indica inciting Karnal bunt disease of wheat.

Tilletia indica incites Karnal bunt (KB) disease in wheat. To date, no KB resistant wheat cultivar could be developed due to non-availability of potential biomarkers related to pathogenicity/virulence for screening of resistant wheat genotypes. The present study was carried out to compare the proteomes of T. indica highly (TiK) and low (TiP) virulent isolates. Twenty one protein spots consistently observed as up-regulated/differential in the TiK proteome were selected for identification by MALDI-TOF/TOF. Identified sequences showed homology with fungal proteins playing essential role in plant infection and pathogen survival, including stress response, adhesion, fungal penetration, invasion, colonization, degradation of host cell wall, signal transduction pathway. These results were integrated with T. indica genome sequence for identification of homologs of candidate pathogenicity/virulence related proteins. Protein identified in TiK isolate as malate dehydrogenase that converts malate to oxaloacetate which is precursor of oxalic acid. Oxalic acid is key pathogenicity factor in phytopathogenic fungi. These results were validated by GC-MS based metabolic profiling of T. indica isolates indicating that oxalic acid was exclusively identified in TiK isolate. Thus, integrated omics approaches leads to identification of pathogenicity/virulence factor(s) that would provide insights into pathogenic mechanisms of fungi and aid in devising effective disease management strategies.

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September 22, 2019

Genomic analyses of unique carbohydrate and phytohormone metabolism in the macroalga Gracilariopsis lemaneiformis (Rhodophyta).

Red algae are economically valuable for food and in industry. However, their genomic information is limited, and the genomic data of only a few species of red algae have been sequenced and deposited recently. In this study, we annotated a draft genome of the macroalga Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta).The entire 88.98 Mb genome of Gp. lemaneiformis 981 was generated from 13,825 scaffolds (=500 bp) with an N50 length of 30,590 bp, accounting for approximately 91% of this algal genome. A total of 38.73 Mb of scaffold sequences were repetitive, and 9281 protein-coding genes were predicted. A phylogenomic analysis of 20 genomes revealed the relationship among the Chromalveolata, Rhodophyta, Chlorophyta and higher plants. Homology analysis indicated phylogenetic proximity between Gp. lemaneiformis and Chondrus crispus. The number of enzymes related to the metabolism of carbohydrates, including agar, glycoside hydrolases, glycosyltransferases, was abundant. In addition, signaling pathways associated with phytohormones such as auxin, salicylic acid and jasmonates are reported for the first time for this alga.We sequenced and analyzed a draft genome of the red alga Gp. lemaneiformis, and revealed its carbohydrate metabolism and phytohormone signaling characteristics. This work will be helpful in research on the functional and comparative genomics of the order Gracilariales and will enrich the genomic information on marine algae.

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September 22, 2019

Genome-wide analysis of the NAC transcription factor family and their expression during the development and ripening of the Fragaria × ananassa fruits.

NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.

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September 22, 2019

Inpactor, integrated and parallel analyzer and classifier of LTR retrotransposons and its application for pineapple LTR retrotransposons diversity and dynamics.

One particular class of Transposable Elements (TEs), called Long Terminal Repeats (LTRs), retrotransposons, comprises the most abundant mobile elements in plant genomes. Their copy number can vary from several hundreds to up to a few million copies per genome, deeply affecting genome organization and function. The detailed classification of LTR retrotransposons is an essential step to precisely understand their effect at the genome level, but remains challenging in large-sized genomes, requiring the use of optimized bioinformatics tools that can take advantage of supercomputers. Here, we propose a new tool: Inpactor, a parallel and scalable pipeline designed to classify LTR retrotransposons, to identify autonomous and non-autonomous elements, to perform RT-based phylogenetic trees and to analyze their insertion times using High Performance Computing (HPC) techniques. Inpactor was tested on the classification and annotation of LTR retrotransposons in pineapple, a recently-sequenced genome. The pineapple genome assembly comprises 44% of transposable elements, of which 23% were classified as LTR retrotransposons. Exceptionally, 16.4% of the pineapple genome assembly corresponded to only one lineage of the Gypsy superfamily: Del, suggesting that this particular lineage has undergone a significant increase in its copy numbers. As demonstrated for the pineapple genome, Inpactor provides comprehensive data of LTR retrotransposons’ classification and dynamics, allowing a fine understanding of their contribution to genome structure and evolution. Inpactor is available at https://github.com/simonorozcoarias/Inpactor.

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September 22, 2019

Isolation and characterization of Bacillus sp. GFP-2, a novel Bacillus strain with antimicrobial activities, from Whitespotted bamboo shark intestine.

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to our society. Natural products from microorganism are regarded as the important substitution antimicrobial agents of antibiotics. We isolated a new strain, Bacillus sp. GFP-2, from the Chiloscyllium plagiosum (Whitespotted bamboo shark) intestine, which showed great inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria. Additionally, the growth of salmon was effectively promoted when fed with inactivated strain GFP-2 as the inhibition agent of pathogenic bacteria. The genes encoding antimicrobial peptides like LCI, YFGAP and hGAPDH and gene clusters for secondary metabolites and bacteriocins, such as difficidin, bacillibactin, bacilysin, surfactin, butirosin, macrolactin, bacillaene, fengycin, lanthipeptides and LCI, were predicted in the genome of Bacillus sp. GFP-2, which might be expressed and contribute to the antimicrobial activities of this strain. The gene encoding ß-1,3-1,4-glucanase was successfully cloned from the genome and this protein was detected in the culture supernatant of Bacillus sp. GFP-2 by the antibody produced in rabbit immunized with the recombinant ß-1,3-1,4-glucanase, indicating that this strain could express ß-1,3-1,4-glucanase, which might partially contribute to its antimicrobial activities. This study can enhance a better understanding of the mechanism of antimicrobial activities in genus Bacillus and provide a useful material for the biotechnology study in antimicrobial agent development.

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September 22, 2019

Nucleotide-binding resistance gene signatures in sugar beet, insights from a new reference genome.

Nucleotide-binding (NB-ARC), leucine-rich-repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long-read sequencing, are improving resolution of repeat-rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB-ARC domain as a model, 231 tentative NB-ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB-ARC signatures as well as full-length sequences. The NB-ARC-associated proteins contained NLR resistance gene domains, including TIR, CC, and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB-ARC domains to validated R genes from monocots and eudicots suggested extensive B. vulgaris-specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR-like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic-acid-based method for NLR prediction in non-model plant species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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