Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetecte

Comparison and transfer testing of multiplex ligation detection methods for GM plants

<p>Abstract</p> <p>Background</p> <p>With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.</p> <p>Results</p> <p>Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).</p> <p>Conclusions</p> <p>From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.</p

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogensCladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu \u3e61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis, causal agent of the black leaf streak disease of banana.

Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2-2 and MfEcp2-3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf-4 and Cf-Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co-evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South-East Asia relative to strains from other banana-producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian-Pacific region. Furthermore, transient co-expression of the MfAvr4 effector alleles and the tomato Cf-4 resistance gene, as well as of MfEcp2, MfEcp2-2 and MfEcp2-3 and the putative Cf-Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field

Novel Introner-Like Elements in fungi Are Involved in Parallel Gains of Spliceosomal Introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom

Detection of Introner-Like Elements (ILEs) in closely related dothideomycetous fungal species.

<p>(A) Maximum-likelihood phylogenetic tree using ITS and LSU sequences. Fungal species in which ILEs have been previously identified are highlighted in bold. <i>Aspergillus niger</i> belongs to the <i>Eurotiomycetes</i> and serves to root the tree. Accession numbers of fungal species from the CBS-KNAW collection are indicated in between brackets. The scale bar indicates the number of substitutions per site. (B) PCR with primers specific to single (<i>cf01</i> and <i>cf02</i>) or shared (<i>cf02cf03ds01ds05</i>, <i>cf04ds03</i> and <i>cf08ds04</i>) ILE families between <i>F</i>. <i>fulva</i> and <i>Dothistroma septosporum</i> was performed using genomic DNA. PCR products were run on 20% acrylamide gels. The first row shows a 50 bp-step DNA ladder and the last row shows the water control. Asterisks indicate fragments that correspond to two different ILEs as revealed by sequencing.</p

Analysis of intron positions in genes with recent insertions of Introner-Like Elements (ILE).

<p>ILEs from <i>Passalora brachycarpa</i> inserted in genes encoding (A) a transporter and (B) a peroxidase. ILEs from <i>Passalora miurae</i> inserted in genes encoding (C) a hydroxylase/oxidoreductase and (D) a fungal transcription factor. For each gene, a maximum likelihood phylogenetic tree was constructed with the predicted protein sequence of orthologues. The trees were rooted with the closest homologue found in Basidiomycota. Bootstrap values of 100 repeats are shown. Scale bar represents the number of substitutions per site. The numbers correspond to the protein ID from the Joint Genome Institute mycocosm portal, except for one gene that was not predicted in the <i>Fulvia fulva</i> genome and for which genomic coordinates are given (B). Orders in fungal classification are mentioned in the trees. On the right, diagrams depict aligned protein sequences and intron positions are indicated as black bars. Their positions in the protein alignment that served to build the phylogenetic trees are indicated above. Positions that are shown in grey highlight putative intron sliding. The black arrows indicate the positions where ILEs inserted in the genes of <i>P</i>. <i>miurae</i> or <i>P</i>. <i>brachycarpa</i>. The open triangles indicate previously identified ILEs. Dots indicate positions where parallel intron gains have occurred. The black bar below each protein representation indicates conserved domains (positions in the protein alignments are indicated between brackets). Asterisks behind species names indicate genes that are likely pseudogenes because of an in frame stop codon. (E) Schematic overview of intron positions (numbered on top) in three genes. The first row shows intron positions in genes of <i>P</i>. <i>miurae</i> or <i>P</i>. <i>brachycarpa</i>. Thick lines indicate monophyletic clades according to the phylogenetic trees. Black, dark grey and light grey squares indicate single presence of an intron position in a monophyletic clade, presence-absence polymorphism and single absence of an intron position in a monophyletic clade, respectively. White squares indicate absence of intron. The presence of ILE and occurrence of putative intron splicing (IS) are indicated below each scheme. <i>Aciri</i>: <i>Acidomyces richmondensis</i>; <i>Altbr</i>: <i>Alternaria brassicicola</i>; <i>Cerzm</i>: <i>Cercospora zeae-maydis</i>; <i>Fulfu</i>: <i>Fulvia fulva</i>; <i>Bipze</i>: <i>Bipolaris zeicola</i>; <i>Bipma</i>: <i>Bipolaris maydis</i>; <i>Curlu</i>: <i>Curvularia lunata</i>; <i>Bipor</i>: <i>Bipolaris oryzae</i>; <i>Bipso</i>: <i>Bipolaris sorokiniana</i>; <i>Bipvi</i>: <i>Bipolaris victoriae</i>; <i>Didex</i>: <i>Didymella exigua</i>; <i>Dotse</i>: <i>Dothistroma septosporum</i>; <i>Lentfl</i>: <i>Lentithecium fluviatile</i>; <i>Pleli</i>: <i>Plenodomus lingam</i>; <i>Lopma</i>: <i>Lophiostoma macrostomum</i>; <i>Psefi</i>: <i>Pseudocercospora fijiensis</i>; <i>Pyrtr</i>: <i>Pyrenophora tritici-repentis</i>; <i>Pyrtt</i>: <i>Pyrenophora teres f</i>. <i>teres</i>; <i>Sphmu</i>: <i>Sphaerulina musiva</i>; <i>Sphpo</i>: <i>Sphaerulina populicola</i>; <i>Parno</i>: <i>Parastagonospora nodorum</i>; <i>Zasce</i>: <i>Zasmidium cellare</i>; <i>Zymtr</i>: <i>Zymoseptoria tritici</i>; <i>Aspve</i>: <i>Aspergillus versicolor</i>; <i>Aspwe</i>: <i>Aspergillus wentii</i>; <i>Penbi</i>: <i>Penicillium bilaiae</i>; <i>Penbr</i>: <i>Penicillium brevicompactum</i>; <i>Penca</i>: <i>Penicillium canescens</i>; <i>Pench</i>: <i>Penicillium chrysogenum</i>; <i>Penfe</i>: <i>Penicillium fellutanum</i>; <i>Pengl</i>: <i>Penicillium glabrum</i>; <i>Penox</i>: <i>Penicillium oxalicum</i>; <i>Colgr</i>: <i>Colletotrichum graminicola</i>; <i>Colfi</i>: <i>Colletotrichum fiorinae</i>; <i>Colgl</i>: <i>Colletotrichum gloeosporioides</i>; <i>Melva</i>: <i>Meliniomyces variabilis</i>; <i>Glotr</i>: <i>Gloeophyllum trabeum</i>; <i>Phlgi</i>: <i>Phlebiopsis gigantean</i>; <i>Psean</i>: <i>Pseudozyma antarctica</i>; <i>Psehu</i>: <i>Pseudozyma hubeiensis</i>.</p

Alignments of newly discovered Introner-Like Elements (ILEs).

<p>DNA sequences obtained from the PCR fragments were aligned, using as references the consensus sequence and the four most conserved ILE sequences of the corresponding families in <i>F</i>. <i>fulva</i> and <i>Dothistroma septosporum</i>. The complete sequences of known ILEs amplified by PCR in this study are also included. Alignments are shown for ILEs related to (A) <i>cf01</i>, (B) <i>cf02cf03ds01ds05</i> and (C) <i>cf08ds04</i> families. Bars above the alignments indicate the oligonucleotide sequences. <i>Fulful</i>: <i>Fulvia fulva</i>; <i>Dotsep</i>: <i>Dothistroma septosporum</i>; <i>Amyafr</i>: <i>Amycosphaerella africana</i>; <i>Pasbra</i>: <i>Passalora brachycarpa</i>; <i>Pascap</i>: <i>Passalora capsicicola</i>; <i>Pasdal</i>: <i>Passalora daleae</i>; <i>Pasmic</i>: <i>Passalora microsora</i>; <i>Pasmiu</i>: <i>Passalora miurae</i>; <i>Pasper</i>: <i>Passalora perfoliati</i>; <i>Passmi</i>: <i>Passalora smilacis</i>.</p

Transcriptome analysis of potato tubers - effects of different agricultural practices

The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments