Sébastien Theil

Structural and functional partitioning of bread wheat chromosome 3B

We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits.

TriAnnot: A Versatile and High Performance Pipeline for the Automated Annotation of Plant Genomes

In support of the international effort to obtain a reference sequence of the bread wheat genome and to provide plant communities dealing with large and complex genomes with a versatile, easy-to-use online automated tool for annotation, we have developed the TriAnnot pipeline. Its modular architecture allows for the annotation and masking of transposable elements, the structural, and functional annotation of protein-coding genes with an evidence-based quality indexing, and the identification of conserved non-coding sequences and molecular markers. The TriAnnot pipeline is parallelized on a 712 CPU computing cluster that can run a 1-Gb sequence annotation in less than 5 days. It is accessible through a web interface for small scale analyses or through a server for large scale annotations. The performance of TriAnnot was evaluated in terms of sensitivity, specificity, and general fitness using curated reference sequence sets from rice and wheat. In less than 8 h, TriAnnot was able to predict more than 83% of the 3,748 CDS from rice chromosome 1 with a fitness of 67.4%. On a set of 12 reference Mb-sized contigs from wheat chromosome 3B, TriAnnot predicted and annotated 93.3% of the genes among which 54% were perfectly identified in accordance with the reference annotation. It also allowed the curation of 12 genes based on new biological evidences, increasing the percentage of perfect gene prediction to 63%. TriAnnot systematically showed a higher fitness than other annotation pipelines that are not improved for wheat. As it is easily adaptable to the annotation of other plant genomes, TriAnnot should become a useful resource for the annotation of large and complex genomes in the future.

Complete Nucleotide Sequence of Artichoke latent virus Shows it to be a Member of the Genus Macluravirus in the Family Potyviridae

Complete genomic sequences of Artichoke latent virus (ArLV) have been obtained by classical or high-throughput sequencing for an ArLV isolate from Italy (ITBr05) and for two isolates from France (FR37 and FR50). The genome is 8,278 to 8,291 nucleotides long and has a genomic organization comparable with that of Chinese yam necrotic mosaic virus (CYNMV), the only macluravirus fully sequenced to date. The cleavage sites of the viral polyprotein have been tentatively identified by comparison with CYNMV, confirming that macluraviruses are characterized by the absence of a P1 protein, a shorter and N-terminally truncated coat protein (CP). Sequence comparisons firmly place ArLV within the genus Macluravirus, and confirm previous results suggesting that Ranunculus latent virus (RALV), a previously described Macluravirus sp., is very closely related to ArLV. Serological relationships and comparisons of the CP gene and of the partial RaLV sequence available all indicate that RaLV should not be considered as a distinct species but as a strain of ArLV. The results obtained also suggest that the spectrum of currently used ArLV-specific molecular hybridization or polymerase chain reaction detection assays should be improved to cover all isolates and strains in the ArLV species.

Determination of the complete genomic sequence of grapevine virus H, a novel vitivirus infecting grapevine

The present work reports the discovery and the complete genome sequencing of a novel member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae) from a symptomless grapevine of unknown variety from Portugal. Total RNAs extracted from phloem scrapings were sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed a mixed infection involving three viruses and two viroids in addition to a novel vitivirus. Completion and analysis of the genome sequence (7446 nt excluding the polyA tail) showed a typical vitivirus genomic organization. Phylogenetic analysis of the various ORFs clearly showed the new virus to belong in the genus Vitivirus, but sequence divergence firmly establishes it as a member of a new species, for which the name “Grapevine virus H” is proposed.

First Report of Nectarine stem pitting-associated virus Infecting Prunus mume in Japan

Nectarine stem pitting-associated virus (NSPaV) is a recently described member of the genus Luteovirus in the family Luteoviridae on which very limited information is available. Initially described in the United States on post-quarantine imported nectarine (Prunus persica) trees (Bag et al. 2015), it was later detected on peach and nectarine plants from California (Villamor et al. 2016). Although it was initially described as tentatively associated with severe stem-pitting symptoms (Bag et al. 2015), NSPaV association with disease remains uncertain because it has been detected in some symptomless trees (Villamor et al. 2016). During a survey in 2015, five Japanese apricot (P. mume) samples collected in Japan and showing various virus-like symptoms such as discolorations and spotting on their leaves were individually analyzed by Illumina deep sequencing of cDNA libraries prepared from purified double-stranded RNAs (Candresse et al. 2013). Analysis of the sequence data showed that in one sample, collected in a park in Morioka, seven contigs showed high nucleotide identity values (90 to 98%) with the closely related NSPaV sequences available in GenBank (accession nos. KP638562, KT273409, and KT273410). These contigs totaled 3.4 kb or about 68% of NSPaV genome (NC_027211; Bag et al. 2015) and corresponded to about 0.37% of the total sequencing reads obtained for the sample. The presence of NSPaV in the sample was confirmed by RT-PCR using the NSPaV-F mod 5′-AGACTMAAATTCAATGARAAGA-3′ and NSPaV-R 5′-TTTCACAAATGATAACACAAT-3′ primer pair modified from Bag et al. (2015). The expected 460-bp fragment, corresponding to positions 1646 to 2106 of the genome (RdRp gene), was obtained and, upon direct sequencing (KX853093), shown to share 96.2 to 96.7% nucleotide identity with the NSPaV isolates present in GenBank. To the best of our knowledge, this is the first report of NSPaV presence in Japan and in P. mume, extending the information on both its host range and geographical distribution. The analysis of the high-throughput sequence data obtained on the NSPaV-infected sample showed it to be also infected by Asian prunus virus 1 and thus, no clear conclusion can be drawn on whether NSPaV may be responsible for any specific symptoms in P. mume.

Draft Genome Sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium, Three Species with Equivocal Pathogenic Status for Cattle

ABSTRACT We report here the draft genome sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium. These three species are regularly isolated from bovine clinical specimens, although their role in disease is unclear.

Draft Genome Sequences of Mycoplasma auris and Mycoplasma yeatsii, Two Species of the Ear Canal of Caprinae.

ABSTRACT We report here the draft genome sequences of Mycoplasma auris and Mycoplasma yeatsii, two species commonly isolated from the external ear canal of Caprinae.

Classical and next generation sequencing approaches unravel Bymovirus diversity in barley crops in France

Despite the generalized use of cultivars carrying the rym4 resistance gene, the impact of viral mosaic diseases on winter barleys increased in recent years in France. This change could reflect i) an increased prevalence of the rym4 resistance-breaking pathotype of Barley yellow mosaic virus Y (BaYMV-2), ii) the emergence of rym4 resistance-breaking pathotypes of Barley mild mosaic virus (BaMMV) or iii) the emergence of other viruses. A study was undertaken to determine the distribution and diversity of viruses causing yellow mosaic disease. A collection of 241 symptomatic leaf samples from susceptible, rym4 and rym5 varieties was gathered from 117 sites. The viruses present in all samples were identified by specific RT-PCR assays and, for selected samples, by double-stranded RNA next-generation sequencing (NGS). The results show that BaYMV-2 is responsible for the symptoms observed in varieties carrying the resistance gene rym4. In susceptible varieties, both BaYMV-1 and BaYMV-2 were detected, together with BaMMV. Phylogenetic analyses indicate that the rym4 resistance-breaking ability independently evolved in multiple genetic backgrounds. Parallel analyses revealed a similar scenario of multiple independent emergence events in BaMMV for rym5 resistance-breaking, likely involving multiple amino acid positions in the viral-linked genome protein. NGS analyses and classical techniques provided highly convergent results, highlighting and validating the power of NGS approaches for diagnostics and viral population characterization.

Determination of the complete genomic sequence of Neofusicoccum luteum mitovirus 1 (NLMV1), a novel mitovirus associated with a phytopathogenic Botryosphaeriaceae

Neofusicoccum luteum species belongs to the Botryosphaeriaceae family and is involved in grapevine wood decay diseases. The present study reports the discovery and the molecular characterization of a novel mitovirus infecting this fungus. Double-stranded RNAs were purified from cultivated N. luteum and analysed by next generation sequencing. Using contigs showing BlastX homology with the RNA-dependent RNA polymerase (RdRp) gene of various members of the family Narnaviridae, a single contig of approximately 1.2 kb was constructed. The genomic sequence was completed and phylogenetic analyses indicated that this virus represents a new member of the genus Mitovirus, for which the name of “Neofusicoccum luteum mitovirus 1” is proposed. The genome is 2,389 nucleotides long and, based on the fungal mitochondrial genetic code, it encodes a putative protein of 710 amino acids, homologous to the RdRps of members of the Narnaviridae family. The neofusicoccum luteus mitovirus 1 (NLMV1) RdRp contains the six conserved motifs previously reported for mitoviral RdRps. Our findings represent the first evidence that a mycovirus can infect N. luteum, an important pathogenic fungus of grapevine.

Molecular Characterization of a Novel Species of Capillovirus from Japanese Apricot (Prunus mume)

With the increased use of high-throughput sequencing methods, new viruses infecting Prunus spp. are being discovered and characterized, especially in the family Betaflexiviridae. Double-stranded RNAs from symptomatic leaves of a Japanese apricot (Prunus mume) tree from Japan were purified and analyzed by Illumina sequencing. Blast comparisons of reconstructed contigs showed that the P. mume sample was infected by a putative novel virus with homologies to Cherry virus A (CVA) and to the newly described Currant virus A (CuVA), both members of genus Capillovirus. Completion of the genome showed the new agent to have a genomic organization typical of capilloviruses, with two overlapping open reading frames encoding a large replication-associated protein fused to the coat protein (CP), and a putative movement protein (MP). This virus shares only, respectively, 63.2% and 62.7% CP amino acid identity with the most closely related viruses, CVA and CuVA. Considering the species demarcation criteria in the family and phylogenetic analyses, this virus should be considered as representing a new viral species in the genus Capillovirus, for which the name of Mume virus A is proposed.