Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds

Main steps of the novel RAD-seq protocol.

<p>1–2) sample genomic DNA is digested. The resulting digested DNA fragments are ligated to a P1 adaptor, that presents a biotin group and a 4 bp overhang complementary to BamHI recognition site. 3-4-5) Biotinilated fragments are random sheared to a target size of 300–200 bp, captured using streptavidin beads and ligated to standard barcoded adaptors for 5500 SOLiD Fragment libraries. 6) RAD-seq libraries are amplified and purified before sequencing.</p

Indices of genetic diversity evaluated in cultivated and wild accessions separately.

<p>Indices of genetic diversity evaluated in cultivated and wild accessions separately.</p

SNP-Discovery by RAD-Sequencing in a Germplasm Collection of Wild and Cultivated Grapevines (<i>V</i>. <i>vinifera</i> L.)

<div><p>Whole-genome comparisons of <i>Vitis vinifera</i> subsp. <i>sativa</i> and <i>V</i>. <i>vinifera</i> subsp. <i>sylvestris</i> are expected to provide a better estimate of the valuable genetic diversity still present in grapevine, and help to reconstruct the evolutionary history of a major crop worldwide. To this aim, the increase of molecular marker density across the grapevine genome is fundamental. Here we describe the SNP discovery in a grapevine germplasm collection of 51 cultivars and 44 wild accessions through a novel protocol of restriction-site associated DNA (RAD) sequencing. By resequencing 1.1% of the grapevine genome at a high coverage, we recovered 34K <i>BamHI</i> unique restriction sites, of which 6.8% were absent in the ‘PN40024’ reference genome. Moreover, we identified 37,748 single nucleotide polymorphisms (SNPs), 93% of which belonged to the 19 assembled chromosomes with an average of 1.8K SNPs per chromosome. Nearly half of the SNPs fell in genic regions mostly assigned to the functional categories of metabolism and regulation, whereas some nonsynonymous variants were identified in genes related with the detection and response to environmental stimuli. SNP validation was carried-out, showing the ability of RAD-seq to accurately determine genotypes in a highly heterozygous species. To test the usefulness of our SNP panel, the main diversity statistics were evaluated, highlighting how the wild grapevine retained less genetic variability than the cultivated form. Furthermore, the analysis of Linkage Disequilibrium (LD) in the two subspecies separately revealed how the LD decays faster within the domesticated grapevine compared to its wild relative. Being the first application of RAD-seq in a diverse grapevine germplasm collection, our approach holds great promise for exploiting the genetic resources available in one of the most economically important fruit crops.</p></div

The decay of LD in <i>sativa</i> and <i>sylvestris</i> populations.

<p>Each point represents the median r² value in sequential bins of 10kb against physical position.</p

Number of identified <i>BamHI</i> recognition sites.

<p>Number of identified <i>BamHI</i> recognition sites.</p

number of alignments per sample.

<p>High quality (MapQ > 10) alignments per sample are shown in green, low quality (MapQ < 10) alignments in yellow and unaligned and multiple aligned reads in red.</p

Minor allele frequency (MAF) distribution within cultivated (yellow) and wild (blue) grapevine populations, taking into account all nuclear SNP loci identified through the novel RAD-seq assay.

<p>Minor allele frequency (MAF) distribution within cultivated (yellow) and wild (blue) grapevine populations, taking into account all nuclear SNP loci identified through the novel RAD-seq assay.</p

IRF1 and NF-kB Restore MHC Class I-Restricted Tumor Antigen Processing and Presentation to Cytotoxic T Cells in Aggressive Neuroblastoma

<div><p>Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed <em>in vivo</em>. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.</p> </div

Expression of MHC-I, IRF1 and IRF2 in NB cell lines.

<p>A, flow cytometry analysis of surface MHC-I expression in NB cell lines using W6/32 mAb (grey lines). Shaded histograms, negative controls stained with isotype-matched primary antibody. B, immunoblot analysis of IRF1 and IRF2 in NB cell lines. Equal amounts of whole-cell extracts and nuclear extracts, as indicated, were resolved by SDS-PAGE, immunoblotted and probed with specific antibodies. ERp57 and PCNA were used for normalization. Positive and negative IRF1 and IRF2 controls, as well as densitometric and statistical analysis of WB bands are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046928#pone.0046928.s001" target="_blank">Fig. S1</a>. C, qRT-PCR analysis of mRNAs from different NB cell lines. 18S RNA was used for normalization. Significant differences between the 3 MHC-I-expressing NB cells and the 5 MHC-I-low NB cells of mRNA expression separately averaged were evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046928#s2" target="_blank">Materials and Methods</a>. IRF1: <i>P</i><0,001; IRF2: <i>P</i> = 0,09; ± SD of triplicate assays. Data shown in the panels A to C are representative of at least 3 independent experiments.</p