Alberto Vecchietti

Genetic dissection of aroma volatile compounds from the essential oil of peach fruit: QTL analysis and identification of candidate genes using dense SNP maps

Volatile organic compounds (VOCs) in plants are involved in aroma and pest resistance. These compounds form a complex mixture whose composition is specific to species and often to varieties. Despite their importance as essential factors that determine peach fruit quality, understanding of molecular, genetic, and physiological mechanisms underlying aroma formation is limited. The aim of this study was the identification in peach of quantitative trait loci (QTLs) for fruit VOCs to understand their genetic basis using an F1 population of 126 seedlings deriving from the cross between “Bolero” (B) and “OroA” (O), two peach cultivars differing in their aroma profile. Dense single nucleotide polymorphism (SNP) and SSR maps covering the eight linkage groups of the peach genome were constructed by genotyping with the International Peach SNP Consortium peach SNP array v1, and data for 23 VOCs with high or unknown “odor activity value” were obtained by gas chromatography–mass spectrometry analysis of fruit essential oil in the years 2007 and 2008. A total of 72 QTLs were identified, most consistent in both years. QTLs were identified for the 23 VOCs studied, including three major QTLs for nonanal, linalool, and for p-menth-1-en-9-al stable in both years. Collocations between candidate genes and major QTLs were identified taking advantage of the peach genome sequence: genes encoding two putative terpene synthases and one lipoxygenase (Lox) might be involved in the biosynthesis of linalool and p-menth-1-en-9-al, and nonanal, respectively. Implications for marker-assisted selection and future research on the subject are discussed.

Identification of key odor volatile compounds in the essential oil of nine peach accessions

BACKGROUND Volatile compounds, together with sugars and acids, are the main chemical species determining the characteristic aroma and flavor of food. In peach, more than 100 volatiles have been identified. RESULTS The essential oil of six peach and three nectarine accessions used in Italian breeding programs was obtained by steam distillation, and the volatiles were investigated. A total of 47 known volatiles, two unidentified compounds and nine hydrocarbons were identified, including 12 aldehydes, six alcohols, three acids, three esters, six terpenes, two phenylalanine derivates, two C(13) norisoprenoids, one ketone (C(9)) and 10 lactones. A wide variation in the number of volatiles and in their concentration was observed among the nine accessions. Twenty-one compounds presented odor activity values (OAVs) higher than 1 in at least one of the accessions and were therefore putatively considered as key odorants in the peach volatile composition. CONCLUSION This study reports the identification, quantification and potency, based on the OAVs, of the most important volatile compounds, along with fruit quality characteristics, of nine different peach/nectarine accessions and will help future peach volatile breeding programs for the selection of odor-rich accessions to be used in the development of new improved cultivars.

ESTree db: a Tool for Peach Functional Genomics

BackgroundThe ESTree db http://www.itb.cnr.it/estree/ represents a collection of Prunus persica expressed sequenced tags (ESTs) and is intended as a resource for peach functional genomics. A total of 6,155 successful EST sequences were obtained from four in-house prepared cDNA libraries from Prunus persica mesocarps at different developmental stages. Another 12,475 peach EST sequences were downloaded from public databases and added to the ESTree db. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts and data were collected in a MySQL database. A php-based web interface was developed to query the database.ResultsThe ESTree db version as of April 2005 encompasses 18,630 sequences representing eight libraries. Contig assembly was performed with CAP3. Putative single nucleotide polymorphism (SNP) detection was performed with the AutoSNP program and a search engine was implemented to retrieve results. All the sequences and all the contig consensus sequences were annotated both with blastx against the GenBank nr db and with GOblet against the viridiplantae section of the Gene Ontology db. Links to NiceZyme (Expasy) and to the KEGG metabolic pathways were provided. A local BLAST utility is available. A text search utility allows querying and browsing the database. Statistics were provided on Gene Ontology occurrences to assign sequences to Gene Ontology categories.ConclusionThe resulting database is a comprehensive resource of data and links related to peach EST sequences. The Sequence Report and Contig Report pages work as the web interface core structures, giving quick access to data related to each sequence/contig.

Version VI of the ESTree db: an improved tool for peach transcriptome analysis

BackgroundThe ESTree database (db) is a collection of Prunus persica and Prunus dulcis EST sequences that in its current version encompasses 75,404 sequences from 3 almond and 19 peach libraries. Nine peach genotypes and four peach tissues are represented, from four fruit developmental stages. The aim of this work was to implement the already existing ESTree db by adding new sequences and analysis programs. Particular care was given to the implementation of the web interface, that allows querying each of the database features.ResultsA Perl modular pipeline is the backbone of sequence analysis in the ESTree db project. Outputs obtained during the pipeline steps are automatically arrayed into the fields of a MySQL database. Apart from standard clustering and annotation analyses, version VI of the ESTree db encompasses new tools for tandem repeat identification, annotation against genomic Rosaceae sequences, and positioning on the database of oligomer sequences that were used in a peach microarray study. Furthermore, known protein patterns and motifs were identified by comparison to PROSITE. Based on data retrieved from sequence annotation against the UniProtKB database, a script was prepared to track positions of homologous hits on the GO tree and build statistics on the ontologies distribution in GO functional categories. EST mapping data were also integrated in the database. The PHP-based web interface was upgraded and extended. The aim of the authors was to enable querying the database according to all the biological aspects that can be investigated from the analysis of data available in the ESTree db. This is achieved by allowing multiple searches on logical subsets of sequences that represent different biological situations or features.ConclusionsThe version VI of ESTree db offers a broad overview on peach gene expression. Sequence analyses results contained in the database, extensively linked to external related resources, represent a large amount of information that can be queried via the tools offered in the web interface. Flexibility and modularity of the ESTree analysis pipeline and of the web interface allowed the authors to set up similar structures for different datasets, with limited manual intervention.

Comparative analysis of expressed sequence tags from tissues in ripening stages of peach ( Prunus persica L. Batsch)

Expressed sequence tag (EST) represents a resource for gene discovery, genome annotation and comparative genomics in plants. ESTs were derived by sequencing clones from five libraries created from two different fruit tissues (skin and mesocarp), at four ripening stages (from post-allegation to post-climacteric) in three different genotypes of peach (OroA, Bolero and Suncrest). A total of 10,847 EST sequences were produced (dataset A); in addition, 21,857 peach ESTs (dataset B) were obtained from public databases. Clustering and assembly of both datasets gave 17,858 unigenes. Analysis of the sequences allowed the assignment of a putative function to 70.8% of the ESTs. In order to define the relationship among fruit tissues transcriptome, a gene ontology analysis was performed. Differences among organs and among different maturation stages of the same organs were identified in organelle, signal transducer and antioxidant activity. A distance matrix of pairwise correlation coefficients analysis was applied between the libraries. Shoot appeared to outgroup and our analysis proved to be an efficient tool to parallel and complement gene expression studies (for example, based on microarray analysis). We conducted an analysis of the frequency of genes putatively involved in the metabolism of some volatiles, which pointed to a predominant presence of those transcripts in the skin. The metabolic pathways of esters and lactones were selected for further isolation and cloning of key genes. The EST database is available at the web site www.itb.cnr.it/estree.

Protein profiling and tps23 induction in different maize lines in response to methyl jasmonate treatment and Diabrotica virgifera infestation

Plant responses to herbivore insects involve direct and indirect defense with the production of signal molecules including jasmonic acid (JA) and its derivatives (e.g. methyl jasmonate, MeJA). In maize (Zea mays), root feeding by Diabrotica virgifera larvae activates an indirect defense mechanism, through enthomopathogenic nematodes that are recruited after Terpene Synthase 23 (tps23) upregulation and (E)-β-caryophyllene root emission. In order to gain insight into the correlation between JA signaling and response to Diabrotica attack, we analyzed tps23 expression and protein profiles in maize roots in response to MeJA treatment and insect infestation. Similar to herbivore feeding, MeJA treatment was found to increase tps23 transcript accumulation, with consistent variations for both treatments in maize lines differing in (E)-β-caryophyllene production. Analysis of root protein profiles showed specific alterations leading to the identification of three proteins that were induced by MeJA treatment. We focused on a peroxidase-like protein (Px-like) showing that the corresponding transcripts accumulated in all tested lines. Results show that exogenous application of MeJA upregulates tps23 expression and specifically alters protein patterns in maize roots. Parallel effects on tps23 transcript accumulation were observed upon hormone exposure and insect infestation in different maize lines. In contrast, Px-like transcript profiling showed differences between treatments. These results support the possible involvement of MeJA in mediating the upregulation of tps23 in response to Diabrotica attack.

Expression profiling of genes involved in the formation of aroma in two peach genotypes

The expression profile of flavour-related genes during ripening was investigated in two peach genotypes, Bolero and OroA, which have been selected for their contrasting aroma/ripening behaviour. A new peach microarray containing 4776 oligonucleotide probes corresponding to a set of ESTs specifically enriched in secondary metabolism (μPEACH2.0) was designed to investigate transcriptome changes during three fruit ripening stages, revealing 1807 transcripts differentially expressed within and between the two genotypes. Differences in the expression of genes involved in the biosynthesis of aroma compounds were detected during the ripening process within and between the two genotypes. In particular, a subset of 12 transcripts involved in metabolism of esters, norisoprenoids, phenylpropanoids and lactones, varied in expression during ripening and between Bolero and OroA.