DUGMO: tool for the detection of unknown genetically modified organisms with high-throughput sequencing data for pure bacterial samples.

BACKGROUND: The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. RESULTS: We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with - possibly partial - transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GM Bacillus subtilis showed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild type B. subtilis, no false positive sequences were detected. CONCLUSION: DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers.

Complete genomic sequence of barley (Hordeum vulgare) endornavirus (HvEV) determined by next-generation sequencing.

Endornaviruses are unusual plant-, fungus- and oomycete-infecting viruses with a large, ca 14- to 17-kb linear double-stranded RNA (dsRNA) genome and a persistent lifestyle. The complete genome sequence of an endornavirus from the barley (Hordeum vulgare) Nerz variety was determined from paired Illumina MySeq reads derived from purified dsRNAs. The genome is 14,243 nt long, with 5' and 3' non-coding regions of 207 and 47 nt, respectively. It encodes a single large protein of 4663 amino acids that carries conserved motifs for a methyltransferase, a helicase and an RNA-dependent RNA polymerase. The sequence of Hordeum vulgare endornavirus (HvEV) carries all the hallmarks of a typical member of the genus Endornavirus, with the exception of an UDP-glycosyltransferase motif observed in many, but not all, endornaviral genomes.

Direct Xylella fastidiosa whole genome sequencing from various plant species using targeted enrichment.

A targeted enrichment method was developed to sequence Xylella fastidiosa genomic DNA directly from plant samples. The method was evaluated on various plant species infected with different strains at different levels of contamination. After enrichment, X. fastidiosa genome coverage was above 99.9% for all tested samples.

Targeted MinION sequencing of transgenes.

The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identified, the presence of an unauthorized GMO is suspected. In this context, the aim of this work was to develop a fast and simple method to obtain the sequence of the transgene and of its junction with plant DNA, with the presence of a screening sequence as only prior knowledge. An unauthorized GM petunia, recently found on the French market, was used as template during the development of this new molecular tool. The innovative proposed protocol is based on the circularization of fragmented DNA followed by the amplification of the transgene and of its flanking regions using long-range inverse PCR. Sequencing was performed using the Oxford Nanopore MinION technology and a bioinformatic pipeline was developed.

Overview and detectability of the genetic modifications in ornamental plants.

The market of ornamental plants is extremely competitive, and for many species genetic engineering can be used to introduce original traits of high commercial interest. However, very few genetically modified (GM) ornamental varieties have reached the market so far. Indeed, the authorization process required for such plants has a strong impact on the profitability of the development of such products. Considering the numerous scientific studies using genetic modification on ornamental species of interest, a lot of transformed material has been produced, could be of commercial interest and could therefore be unintentionally released on the market. The unintentional use of GM petunia in breeding programs has indeed recently been observed. This review lists scientific publications using GM ornamental plants and tries to identify whether these plants could be detected by molecular biology tools commonly used by control laboratories.

Fluorescent-based techniques for viral detection, quantification, and characterization.

Fluorescent-based technologies offer opportunities for developing new assays for detection, quantification, and characterization of viral isolates. According to the intrinsic characteristics of fluorescent-based tools (high specificity, sensitivity, and reliability), such type of molecular assays makes possible investigations on original studies such as evolutionary processes (including fitness measurement of isolates), quantitative epidemiology, or the analysis of synergism and antagonism between closely related isolates. The development of these tools is very simple and requires, in complement to basic molecular knowledge such as extraction, cloning, and (RT)-PCR procedures, only the identification of short specific sequence(s) in the targeted viral genome. The Single Nucleotide Polymorphism (SNP) and the 'real-time' RT-PCR assays are proposed as fluorescent-based tools for qualitative and quantitative viral detection, respectively. Moreover, the SNaPshot technology is described as method for isolate characterization.

Analysis of population structures of viral isolates using single-strand conformation polymorphism method.

The analysis of viral populations requires the use of techniques that describe characteristics of individuals. The single-strand conformation polymorphism (SSCP) makes possible the identification of genetic differences between viral sequences and constitutes an alternative to the expensive and time-consuming cloning and sequencing strategies. Applied to small genomic regions (from 100 to 500 bases in length), SSCP patterns could describe, under appropriate experimental conditions, single nucleotide variations in the studied sequence. The different steps of a complete SSCP procedure, from sampling to pattern analysis (including nucleic acid extraction, RT-PCR amplification, double-stranded DNA quantification, polyacrylamide gel preparation, electrophoresis conditions, and staining procedures), are described using a region (500 bases) of the barley yellow dwarfvirus-PAV (BYDV-PAV, Luteovirus) genome as molecular target.

Targeted Next Generation Sequencing to study insert stability in genetically modified plants.

The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. In the present work, a targeted Next-Generation Sequencing (NGS) approach using barcodes to specifically tag each individual DNA molecules during library preparation was implemented to detect mutations taking into account the background noise due to amplification and sequencing errors. The method was first showed to be efficient in detecting the mutations in synthetic samples prepared with custom-synthesized mutated or non-mutated P35S sequences mixed in different proportions. The genetic stability of a portion of the P35S promoter targeted for GM detection was then analyzed in GM flour samples. Several low frequency mutations were detected in the P35S sequences. Some mutated nucleotides were located within the primers and probes used in the P35S diagnostic test. If present not as somatic mutations but as the consensus sequence of some individuals, these mutations could influence the efficiency of the P35S real time PCR diagnostic test. This methodology could be implemented in genetic stability studies of GM inserts but also to detect single nucleotide mutant GM plants produced using "new breeding techniques".

A multiple single nucleotide polymorphisms interrogation assay for reliable Potato virus Y group and variant characterization.

The complex Potato virus Y classification, including groups (PVYN and PVYO) and variants (PVYNTN and PVYN-W), is based mainly on biological properties of isolates. Published PVY detection tools targeting markers not associated with biological properties could fail to assign correctly isolates in the current classification. To improve PVY detection tools, a single nucleotide polymorphism (SNaPshot) detection assay was developed. The technique was adapted to target the T/C9259, A/C2271, G/C8573 and A/G2213 PVY polymorphic nucleotides. The "TAGA", "CCCG", "CACA" and "CAGA" four-digit codes associated with tested samples allowed identification of PVYN, PVYO, PVYN-W and PVYNTN isolates, respectively. The PVY SNaPshot procedure is efficient and reliable for PVY detection and characterization in samples containing as few as 10(2) viral RNA copies. Moreover, PVY group assignment is possible for fractions containing only 10 copies of a PVY RNA genome. Finally, the SNaPshot assay allows PVY(N)/PVYO dual characterization for mixed samples containing PVYN/PVYO quantity ratios in the range of 0.1-10. This innovative SNaPshot tool improved clearly PVY diagnostic assays described previously by targeting simultaneously major functional markers and sequence unlinked to biological properties used separately in PVY detection tools available currently.

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.

A dCAPS assay detects and characterizes BaYMV according to its ability to overcome rym4-mediated resistance.

Winter barley is subjected to severe yield losses due to the yellow mosaic virus disease. Two soil borne bymoviruses, BaYMV (Barley yellow mosaic virus) and BaMMV (Barley mild mosaic virus) are responsible for this disease in Europe. As breeding resistant cultivars is the only control method against the disease, barley varieties carrying the recessive resistance rym4 were introduced. However, a new pathotype BaYMV-2 overcoming rym4 resistance appeared in the late 1980s. In France, little is known about BaYMV-2 and the common BaYMV (BaYMV-1) distribution, but the increase of the disease occurrence is becoming a concern. There is currently no valid molecular tool for BaYMV-1 and BaYMV-2 differentiation; thus the development of a dCAPS (derived Cleaved Amplified Polymorphic Sequences) tool was investigated. BaYMV-1 and BaYMV-2 diversity was first estimated by Sanger sequencing. The Single Nucleotide Polymorphism (SNP) previously described as providing the ability to overcome rym4-mediated resistance was targeted. A dCAPS tool was developed to digest specifically BaYMV-1. This assay was successfully tested with seventy naturally infected samples. This new tool will be useful to investigate BaYMV-1 and 2 distributions.

Molecular Identification of Broomrape Species from a Single Seed by High Resolution Melting Analysis.

Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.

Complementations and exclusions between mutated versions of a potato virus Y genotype during mixed infections of Nicotiana hosts.

Understanding the processes that have led to the recent prevalence of necrotic genotypes in PVY populations is an important challenge for research programs studying this virus. Non-necrotic PVY(O)-139, necrotic PVY(N)-605 and point mutated versions of PVY(N)-605 (PVY(KRED), PVY(KR) and PVY(ED)), were used in mixtures to inoculate two Nicotiana hosts which express (N. tabacum cv. Xanthi) or not (N. clevelandii) necrosis symptoms in response to infection by PVY(N) group members. The comparison during serial passage experiments of proportions of PVY genotypes produced in mixed infected plants with those of the inocula was used to describe: (i) complementation between PVY(KR) and PVY(N) and between PVY(KRED) and PVY(O) genotypes; (ii) exclusion of the PVY(KRED) genotype, previously described as fitter, during mixed infections in the presence of one of the less fit PVY(N), PVY(ED) and PVY(KR) genotypes and (iii) the prevalence of the non-necrotic PVY(KR) genotype in the presence of PVY(N) parental sequence. These results indicate that the role of both A/G(2213) and A/C(2271) nucleotides in the fitness of PVY genotypes depends on other genetic information in the viral genome that has not yet been identified. Moreover, the collected data indicate that mutation of the nucleotide 2213 in the PVY(N)-605 sequence could lead to the prevalence, both in N. tabacum cv. Xanthi and in N. clevelandii, of the non-necrotic PVY(KR) genotype.

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants.

The prevalence of necrotic potato virus Y (PVY) in natural populations could reflect increased fitness of necrotic isolates. In this paper, the effects of the acquisition of molecular determinants (A/G(2213) and A/C(2271)) involved in necrosis capacity on both the number of progeny produced and the competitiveness of PVY were characterized. The relationship between necrosis and fitness was tested using (i) Nicotiana tabacum cv. Xanthi and Nicotiana clevelandii, (ii) necrotic PVY(N)-605 and non-necrotic PVY(O)-139 isolates, (iii) single-mutated (PVY(KR) and PVY(ED)) and double-mutated (PVY(KRED)) versions of PVY(N)-605 and (iv) three quantitative PCR assays specific for nt A(2213), G(2213) and A(2271) of the PVY genome. The data demonstrated effects of both the genetic background and nt 2213 and 2271 on the fitness of PVY. Quantification of PVY RNA in singly infected plants revealed that both the PVY(N)-605 genetic background and the acquisition of necrotic capacity resulted in a decrease in the number of progeny produced. Competition experiments revealed that the genetic background of PVY(N) had a positive impact on competitiveness. In contrast, nucleotides involved in necrotic properties were associated with decreased fitness. Finally, in the host that did not respond to infection with necrosis, the benefit associated with the PVY(N)-605 genetic background was higher than the cost associated with the acquisition of molecular determinants involved in necrosis capacity. The opposite result was obtained in the host responding to the infection with necrosis. These results indicate that the emergence of necrotic isolates from a non-necrotic population is unlikely in tobacco.