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July 7, 2019  |  

Nanoarrays on passivated aluminum surface for site-specific immobilization of biomolecules

The rapid development of biosensing platforms for highly sensitive and specific detection raises the desire of precise localization of biomolecules onto various material surfaces. Aluminum has been strategically employed in the biosensor system due to its compatibility with CMOS technology and its optical and electrical properties such as prominent propagation of surface plasmons. Herein, we present an adaptable method for preparation of carbon nanoarrays on aluminum surface passivated with poly(vinylphosphonic acid) (PVPA). The carbon nanoarrays were defined by means of electron beam induced deposition (EBID) and they were employed to realize site-specific immobilization of target biomolecules. To demonstrate the concept, selective streptavidin/neutravidin immobilization on the carbon nanoarrays was achieved through protein physisorption with a significantly high contrast of the carbon domains over the surrounding PVPA-modified aluminum surface. By adjusting the fabrication parameters, local protein densities could be varied on similarly sized nanodomains in a parallel process. Moreover, localization of single 40 nm biotinylated beads was achieved by loading them on the neutravidin-decorated nanoarrays. As a further demonstration, DNA polymerase with a streptavidin tag was bound to the biotin-beads that were immobilized on the nanoarrays and in situ rolling circle amplification (RCA) was subsequently performed. The observation of organized DNA arrays synthesized by RCA verified the nanoscale localization of the enzyme with retained biological activity. Hence, the presented approach could provide a flexible and universal avenue to precise localizing various biomolecules on aluminum surface for potential biosensor and bioelectronic applications.

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July 7, 2019  |  

Genome size estimation of Chinese cultured artemisia annua L.

Almost all of antimalarial artemisinin is extracted from the traditional Chinese medicinal plant Artemisia annua L. However, under the condition of insufficient genomic in- formation and unresolved genetic backgrounds, regulatory mechanism of artemisinin biosynthetic pathway has not yet been clear. The genome size of genuine A. annua plants is an especially important and fundamental parameter, which helpful for further insight into genomic studies of ar- temisinin biosynthesis and improvement. In current study, all those genome sizes of A. annua samples collected with Barcoding identification were evaluated to be 1.38-1.49 Gb by Flow Cytometry (FCM) with Nipponbare as the bench- mark calibration standard and soybean and maize as two internal standards individually and simultaneously. The ge- nome estimation of seven A. annua strains came from five China provinces (Shandong, Hunan, Chongqing, Sichuan, and Hainan) with a low coefficient of variation (CV, = 2.96%) wasrelative accurate, 12.87% (220 Mb) less than previous reports about a foreign A. annuaspecies with a single con- trol. It facilitated the schedule of A. annua whole genome sequencing project, optimization of assembly methods and insight into its subsequent genetics and evolution.

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July 7, 2019  |  

Recent advances on detection and characterization of fruit tree viruses using high-throughput sequencing technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

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July 7, 2019  |  

Complete genome sequence of herpes simplex virus 2 strain 333.

Herpes simplex virus 2, or human herpesvirus 2, is a ubiquitous human pathogen that causes genital ulcerations and establishes latency in sacral root ganglia. We fully sequenced and manually curated the viral genome sequence of herpes simplex virus 2, strain 333 using Pacific Biosciences and Illumina sequencing technologies.

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July 7, 2019  |  

Emergence of gyrovirus 3 in commercial broiler chickens with transmissible viral proventriculitis.

Gyrovirus 3 (GyV3) has been identified in faeces from children with acute gastroenteritis. However, whether GyV3 is prevalent in poultry has not been determined to date. To the best of our knowledge, this study is the first to isolate GyV3 from commercial broiler chickens with transmissible viral proventriculitis (TVP) in China. The complete genome of the virus shares 98.4% sequence identity with the FecGy strain that causes acute gastroenteritis in children. Epidemiological investigation from 2013 to 2017 revealed that the infection rate of GyV3 reached 12.5% (42/336) in commercial broiler chickens with TVP, indicating that the infection of GyV3 was ubiquitous in chickens. The emergence of GyV3 in commercial broiler chickens should be highly concerning for public health.© 2018 Blackwell Verlag GmbH.

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July 7, 2019  |  

Complete genome sequences of three Leptospira mayottensis strains from tenrecs that are endemic in the Malagasy region

Leptospirosis is a zoonosis caused by Leptospira, a diversified genus containing more than 10 pathogenic species. Tenrecs are small terrestrial mammals endemic in the Malagasy region and are known to be reservoirs of the recently de- scribed species Leptospira mayottensis. We report the complete genome sequences of three L. mayottensis strains isolated from two tenrec species.

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July 7, 2019  |  

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

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July 7, 2019  |  

Bridging gaps in transposable element research with single-molecule and single-cell technologies

More than half of the genomic landscape in humans and many other organisms is composed of repetitive DNA, which mostly derives from transposable elements (TEs) and viruses. Recent technological advances permit improved assessment of the repetitive content across genomes and newly developed molecular assays have revealed important roles of TEs and viruses in host genome evolution and organization. To update on our current understanding of TE biology and to promote new interdisciplinary strategies for the TE research community, leading experts gathered for the 2nd Uppsala Transposon Symposium on October 4–5, 2018 in Uppsala, Sweden. Using cutting-edge single-molecule and single-cell approaches, research on TEs and other repeats has entered a new era in biological and biomedical research.

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July 7, 2019  |  

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-ß were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-a were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

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July 7, 2019  |  

De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

Long-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.

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