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Grapevine (Vitis vinifera L.) is one of the major fruit crop worldwide with thousands of cultivated varieties. The economical success of the grape and wine related agribusiness depends on the quantity and quality of the grapes produced, which are heavily influenced by genotype and environment. Among the important Italian varieties, 'Nebbiolo' is a valuable cultivar from Piedmont renowned for its use in producing high quality red wines (Barolo, Barbaresco and others). 'Nebbiolo' is characterized by a wide genetic variability expressed by a high number of clones (i.e. vegetatively propagated lines of selected mother plants) registered in the National Catalogue. However, little morphological differences exist between clones and often it is very difficult to visually distinguish the different clones. Furthermore, an important point is to ensure traceability of the vine supply chain, from grape nurseries up to wine industry, in order to provide a product of sure origin to the consumers. To study clonal diversity, simple sequence repeats (SSRs) have been used as markers, however with low success rates in discriminating clones. Recently, studies reported that allelic variations within the genome of a plant species could be identified by using single nucleotide polymorphisms (SNPs) as high-throughput sequence-based markers. The aim of this on-going study is to identify a set of SNPs able to discriminate the registered 'Nebbiolo' clones, setting up an effective and rapid SNP genotyping protocol based on the TaqMan® probes technology. Three 'Nebbiolo' clones were sequenced by Illumina technology (CVT 71, CVT 185 and CVT 423) and processed reads were mapped against the PN40024 reference genome. SNPs were first detected using the GATK-UnifiedGenotyper tool with default parameters followed by filtering and by a calibration step. Moreover, for the allelic variations identified in coding regions, prediction of changes in the amino acid sequence was performed using SNPeff tool. Then, SNPs were validated as true positive by Sanger sequencing and divided in: i) clone specific SNPs, ii) clones common SNPs, iii) SNPs in protein coding region by affecting genes. To date, Sanger sequencing confirmed 11 SNPs (29%), and nine of these were successfully used to perform TaqMan® genotyping assays, amplifying and detecting specific SNP alleles in genomic DNA from 37 'Nebbiolo' clones. Combining results obtained from each single SNP assay, we classified the 'Nebbiolo' clones in six main groups linked to the geographical origin of accessions. This work is in progress to implement the set of SNPs and to improve 'Nebbiolo' discrimination from other grape cultivars. Furthermore, SNPs validation of allelic variations in coding regions involved in relevant biochemical pathways (i.e., flavonoid biosynthesis) is in progress as well.

Introduction. Mutations, chromosomal rearrangements and associated gene fusions resulting from inversions, interstitial deletion or translocations represent recurrent findings in leukemia. Nowadays high-throughput “Next Generation Sequencing” technologies and novel developed algorithmic methods allow a unified deep analysis of such alterations with a single base resolution. Taking advantage from these tools, we performed an RNA-Seq (whole transcriptome shotgun sequencing) approach using the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations and fusion genes in 3 adult BCR-ABL1+ ALL cases treated with tyrosine kinase inhibitors. Methods. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case with del(9)(p13p22). All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. Results. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. Thereafter, we analyzed RNA-seq data by deFuse (McPherson A et al. PLoS Computational Biology May 2011), a novel computational method for fusion discovery between a gene and an intergenic region. This software uses clusters of discordant paired-end alignments to inform a split read alignment analysis for finding fusion boundaries. First of all, we evaluated the ability of deFuse to rediscovery the BCR-ABL1 gene fusion, then we focused on the most intriguing fusions such as the PBXIP1- PMVK (pre-B-cell leukemia homeobox interacting protein 1- phosphomevalonate kinase) gene fusion. All new gene fusions are under evaluation in a bigger ALL cohort. Conclusions. This study provided a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations and gene fusions involved in Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna- Ravenna, FIRB 2006, Ateneo RFO grants, PIO project, Programma di Ricerca Regione–Università 2007–2009.

Nowadays high throughput “Next Generation Sequencing” technologies are revolutionizing genomics and transcriptomics by providing a cost-efficient and single base resolution tool for a unified deep analysis of tumor complexity. Taking advantage from this tool, we used the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations in 3 adult BCRABL1+ ALL cases treated with tyrosine kinase inhibitors. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case. All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. A functional analysis was carried out on common mutated genes using the GeneGo software (www.genego.com) and in the list of the most significant GeneGo Pathway Maps we found the “Development Hedgehog Signaling” (p value = 1.400e-4), including genes such as casein kinase 1, alpha 1-like (CSNK1A1L), catenin (cadherin-associated protein) beta 1 (CTNNB1) and heat shock protein HSP 90-beta (HSP90AB1). In conclusion, this study provided, for the first time, a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations potentially involved in ALL. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione–Università 2007–2009.

Introduction. The BCR-ABL1+ ALL is the most frequent and prognostically unfavorable subtype of ALL in adults. In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. The selected cases had previously been profiled by high-resolution SNP and gene expression arrays and candidate gene re-sequencing. Methods. Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results. RNA-seq generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, most of which successfully mapped to the reference sequence of the human genome. With the exclusion of the T315I BCR-ABL mutation, 7 novel missense mutations were detected after applying stringent criteria to reduce the SNV discovery false positive rate: 4 were exclusively found in the primary ALL sample and affected genes involved in metabolic processes (DPEP1, ZC3H12D, TMEM46) or transport (MVP); 3 relapse-related mutations affected genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21). Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during progression. Moreover, 4,334 and 3,651 primary and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. Finally, a detailed gene expression profile was obtained indicating that more than 60% of annotated human genes were transcribed in leukemia cells in both diagnosis and relapse phases. Approximately 23% of genes were up-regulated at relapse compared to diagnosis, and most of them affected cell cycle progression (AURORA A, SURVIVIN, PLK1, CDK1, Cyclin A, Cyclin B), suggesting that the loss of cell cycle control may play a role in disease progression. Conversely, only 9% of active genes in both samples were down-regulated at relapse compared to diagnosis. Conclusions. Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna,FIRB 2006, PRIN 2008, European LeukemiaNet, GIMEMA ONLUS, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

Nebbiolo is a valuable Vitis vinifera cultivar from Piedmont, north-west Italy, and it is renowned for its use in producing high quality red wines. Very little information is available so far for this cultivar at molecular genetics or genomics level. In this work we present the assembly and the functional characterization of the genome of three clonal selections of the Nebbiolo variety. Genomic DNA libraries from Nebbiolo Michet (clone 71), Nebbiolo Picotener (clone 423) and Nebbiolo Lampia (clone 185) were deep sequenced using an Illumina HiSeq 1000 sequencer. A genomic coverage of 60x was achieved for all libraries. The three genomes were assembled using a bioinformatic pipeline which combines a reference-guided iterative mapping of the sequenced reads with a de novo assembly. Computational analysis showed differences ranging from 1.78 to 1.91 x 106 single nucleotide polymorphisms (SNPs) between the Nebbiolo clones and the Vitis vinifera PN40024 reference genome. Transposable elements, already found in genomes of other plants as well as of other V. vinifera, cover a fraction of more than 34% of the Nebbiolo genomes. A pool of RNA isolated from Nebbiolo plant organs at different developmental stages was deep sequenced and the obtained reads were used to re-annotate the three genomes. The results of this work will open a path for a better understanding of the Nebbiolo genetic variability and the interaction between environmental factors and genotype, along with the possible identification of molecular markers able to distinguish different 'Nebbiolo' clones. This research was funded by Fondazione Cassa di Risparmio di Cuneo, Project "NEBBIOLO GENOMICS: genomica strutturale e funzionale su aspetti patologici e qualitativi"

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (K (d) = 4.0 x 10(-2) mum, at pH 7.0 and 25 degreesC), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana . In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.

Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been applied rarely because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of both transcriptomes, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in Petunia axillaris and Petunia inflata and to explore the molecular basis of the seed coat defects in a Petunia hybrida mutant, anthocyanin 11 (an11), lacking a WD40-repeat (WDR) transcription regulator. Among the transcripts differentially expressed in an11 seeds compared with wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida. © 2011 Blackwell Publishing Ltd.

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Here, for the first time, a comprehensive transcriptomics study is presented of leaf senescence in the legume model Medicago truncatula, providing a broad overview of differentially expressed transcripts involved in this process. The cDNA-amplification fragment length polymorphism (AFLP) technique was used to identify > 500 genes, which were cloned and sorted into functional categories according to their gene ontology annotation. Comparison between the datasets of Arabidopsis and M. truncatula leaf senescence reveals common physiological events but differences in the nitrogen metabolism and in transcriptional regulation. In addition, it was observed that a minority of the genes regulated during leaf senescence were equally involved in other processes leading to programmed cell death, such as nodule senescence and nitric oxide signalling. This study provides a wide transcriptional profile for the comprehension of key events of leaf senescence in M. truncatula and highlights a possible regulative role for MADS box transcription factors in the terminal phases of the process