Paola Tononi

Identification of Putative Stage-Specific Grapevine Berry Biomarkers and Omics Data Integration into Networks

The analysis of grapevine (Vitis vinifera) berries at the transcriptomic, proteomic, and metabolomic levels can provide great insight into the molecular events underlying berry development and postharvest drying (withering). However, the large and very different data sets produced by such investigations are difficult to integrate. Here, we report the identification of putative stage-specific biomarkers for berry development and withering and, to our knowledge, the first integrated systems-level study of these processes. Transcriptomic, proteomic, and metabolomic data were integrated using two different strategies, one hypothesis free and the other hypothesis driven. A multistep hypothesis-free approach was applied to data from four developmental stages and three withering intervals, with integration achieved using a hierarchical clustering strategy based on the multivariate bidirectional orthogonal projections to latent structures technique. This identified stage-specific functional networks of linked transcripts, proteins, and metabolites, providing important insights into the key molecular processes that determine the quality characteristics of wine. The hypothesis-driven approach was used to integrate data from three withering intervals, starting with subdata sets of transcripts, proteins, and metabolites. We identified transcripts and proteins that were modulated during withering as well as specific classes of metabolites that accumulated at the same time and used these to select subdata sets of variables. The multivariate bidirectional orthogonal projections to latent structures technique was then used to integrate the subdata sets, identifying variables representing selected molecular processes that take place specifically during berry withering. The impact of this holistic approach on our knowledge of grapevine berry development and withering is discussed.

De novo transcriptome characterization of Vitis vinifera cv. Corvina unveils varietal diversity.

BackgroundPlants such as grapevine (Vitis spp.) display significant inter-cultivar genetic and phenotypic variation. The genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors.ResultsWe have shown that cDNA sequencing by RNA-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar (Corvina) and the PN40024 reference genome. We detected 15,161 known genes including 9463 with novel splice isoforms, and identified 2321 potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. We also discovered 180 apparent private genes in the Corvina genome which were missing from the reference genome.ConclusionsThe de novo assembly approach allowed a substantial amount of the Corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. The private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. Therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes.

The High Polyphenol Content of Grapevine Cultivar Tannat Berries Is Conferred Primarily by Genes That Are Not Shared with the Reference Genome

The Tannat grape berry is used to produce high-quality wines with an intense purple color and remarkable antioxidant properties. Through reference-guided assembly of the genome combined with de novo assembly of the transcriptome, we found that the variety-specific genes that might contribute substantially to the unique characteristics of the Tannat berry are not present in the reference genome. The grapevine (Vitis vinifera) cultivar Tannat is cultivated mainly in Uruguay for the production of high-quality red wines. Tannat berries have unusually high levels of polyphenolic compounds, producing wines with an intense purple color and remarkable antioxidant properties. We investigated the genetic basis of these important characteristics by sequencing the genome of the Uruguayan Tannat clone UY11 using Illumina technology, followed by a mixture of de novo assembly and iterative mapping onto the PN40024 reference genome. RNA sequencing data for genome reannotation were processed using a combination of reference-guided annotation and de novo transcript assembly, allowing 5901 previously unannotated or unassembled genes to be defined and resulting in the discovery of 1873 genes that were not shared with PN40024. Expression analysis showed that these cultivar-specific genes contributed substantially (up to 81.24%) to the overall expression of enzymes involved in the synthesis of phenolic and polyphenolic compounds that contribute to the unique characteristics of the Tannat berries. The characterization of the Tannat genome therefore indicated that the grapevine reference genome lacks many genes that appear to be relevant for the varietal phenotype.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short‐arm chromosome 5A (5AS) and long‐arm chromosome 5A (5AL) arm‐specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high‐quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map‐based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Nitrate induction triggers different transcriptional changes in a high and a low nitrogen use efficiency maize inbred line.

In higher plants, NO3(-) can induce its own uptake and the magnitude of this induction is positively related to the external anion concentration. This phenomenon has been characterized in both herbaceous and woody plants. Here, different adaptation strategies of roots from two maize (Zea mays L., ZmAGOs) inbred lines differing in nitrogen use efficiency (NUE) and exhibiting different timing of induction were discussed by investigating NO3(-) -induced changes in their transcriptome. Lo5 line (high NUE) showing the maximum rate of NO3(-) uptake 4 h after the provision of 200 μmol/L NO3(-) treatment modulated a higher number of transcripts relative to T250 (low NUE) that peaked after 12 h. The two inbred lines share only 368 transcripts that are modulated by the treatment with NO3(-) and behaved differently when transcripts involved in anion uptake and assimilation were analyzed. T250 line responded to the NO3(-) induction modulating this group of genes as reported for several plant species. On the contrary, the Lo5 line did not exhibit during the induction changes in this set of genes. Obtained data suggest the importance of exploring the physiological and molecular variations among different maize genotypes in response to environmental clues like NO3(-) provision, in order to understand mechanisms underlying NUE.

DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma

BackgroundAlcohol is a well-known risk factor for hepatocellular carcinoma (HCC), but the mechanisms underlying the alcohol-related hepatocarcinogenesis are still poorly understood. Alcohol alters the provision of methyl groups within the hepatic one-carbon metabolism, possibly inducing aberrant DNA methylation. Whether specific pathways are epigenetically regulated in alcohol-associated HCC is, however, unknown. The aim of the present study was to investigate the genome-wide promoter DNA methylation and gene expression profiles in non-viral, alcohol-associated HCC. From eight HCC patients undergoing curative surgery, array-based DNA methylation and gene expression data of all annotated genes were analyzed by comparing HCC tissue and homologous cancer-free liver tissue.ResultsAfter merging the DNA methylation with gene expression data, we identified 159 hypermethylated-repressed, 30 hypomethylated-induced, 49 hypermethylated-induced, and 56 hypomethylated-repressed genes. Notably, promoter DNA methylation emerged as a novel regulatory mechanism for the transcriptional repression of genes controlling the retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22, RDH16), iron homeostasis (HAMP), one-carbon metabolism (SHMT1), and genes with a putative, newly identified function as tumor suppressors (FAM107A, IGFALS, MT1G, MT1H, RNF180).ConclusionsA genome-wide DNA methylation approach merged with array-based gene expression profiles allowed identifying a number of novel, epigenetically regulated candidate tumor-suppressor genes in alcohol-associated hepatocarcinogenesis. Retinol metabolism genes and SHMT1 are also epigenetically regulated through promoter DNA methylation in alcohol-associated HCC.Due to the reversibility of epigenetic mechanisms by environmental/nutritional factors, these findings may open up to novel interventional strategies for hepatocarcinogenesis prevention in HCC related to alcohol, a modifiable dietary component.

Patchwork sequencing of tomato San Marzano and Vesuviano varieties highlights genome-wide variations

BackgroundInvestigation of tomato genetic resources is a crucial issue for better straight evolution and genetic studies as well as tomato breeding strategies. Traditional Vesuviano and San Marzano varieties grown in Campania region (Southern Italy) are famous for their remarkable fruit quality. Owing to their economic and social importance is crucial to understand the genetic basis of their unique traits.ResultsHere, we present the draft genome sequences of tomato Vesuviano and San Marzano genome. A 40x genome coverage was obtained from a hybrid Illumina paired-end reads assembling that combines de novo assembly with iterative mapping to the reference S. lycopersicum genome (SL2.40). Insertions, deletions and SNP variants were carefully measured. When assessed on the basis of the reference annotation, 30% of protein-coding genes are predicted to have variants in both varieties. Copy genes number and gene location were assessed by mRNA transcripts mapping, showing a closer relationship of San Marzano with reference genome. Distinctive variations in key genes and transcription/regulation factors related to fruit quality have been revealed for both cultivars.ConclusionsThe effort performed highlighted varieties relationships and important variants in fruit key processes useful to dissect the path from sequence variant to phenotype.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.)

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

De novo genome assembly of the soil-borne fungus and tomato pathogen Pyrenochaeta lycopersici

BackgroundPyrenochaeta lycopersici is a soil-dwelling ascomycete pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. Fungal pathogens that infect roots receive less attention than those infecting the aerial parts of crops despite their significant impact on plant growth and fruit production.ResultsWe assembled a 54.9Mb P. lycopersici draft genome sequence based on Illumina short reads, and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. Several gene families related to host–pathogen interactions are strongly represented, including those responsible for nutrient absorption, the detoxification of fungicides and plant cell wall degradation, the latter confirming that much of the genome is devoted to the pathogenic activity of the fungus. We did not find a MAT gene, which is consistent with the classification of P. lycopersici as an imperfect fungus, but we observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI).ConclusionsThe P. lycopersici draft genome sequence provided insight into the molecular and genetic basis of the fungal lifestyle, characterizing previously unknown pathogenic behaviors and defining strategies that allow this asexual fungus to increase genetic diversity and to acquire new pathogenic traits.

Transcriptional Reprogramming of the Mycoparasitic Fungus Ampelomyces quisqualis During the Powdery Mildew Host-Induced Germination.

Ampelomyces quisqualis is a mycoparasite of a diverse range of phytopathogenic fungi associated with the powdery mildew disease. Among them are several Erysiphaceae species with great economic impact on high-value crops such as grape. Due to its ability to parasitize and prevent the spread of powdery mildews, A. quisqualis has received considerable attention for its biocontrol potential. However, and in sharp contrast to the extensively studied biocontrol species belonging to the genus Trichoderma, little is known about the biology of A. quisqualis at the molecular and genetic levels. We present the first genome-wide transcription profiling in A. quisqualis during host-induced germination. A total of 1,536 putative genes showed significant changes in transcription during the germination of A. quisqualis. This finding denotes an extensive transcriptional reprogramming of A. quisqualis induced by the presence of the host. Several upregulated genes were predicted to encode for putative mycoparasitism-related proteins such as secreted proteases, virulence factors, and proteins related to toxin biosynthesis. Our data provide the most comprehensive sequence resource currently available for A. quisqualis in addition to offering valuable insights into the biology of A. quisqualis and its mycoparasitic lifestyle. Eventually, this may improve the biocontrol capacity of this mycoparasite.