Adriana Sacco

The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation

BackgroundHigh levels of ascorbic acid (AsA) in tomato fruits provide health benefits for humans and also play an important role in several aspects of plant life. Although AsA metabolism has been characterized in detail, the genetic mechanisms controlling AsA accumulation in tomatoes are poorly understood. The transcriptional control of AsA levels in fruits can be investigated by combining the advanced genetic and genomic resources currently available for tomato. A comparative transcriptomic analysis of fruit tissues was carried out on an introgression line containing a QTL promoting AsA accumulation in the fruit, using a parental cultivar with lower AsA levels as a reference.ResultsIntrogression line IL 12-4 (S. pennellii in a S. lycopersicum background) was selected for transcriptomic analysis because it maintained differences in AsA levels compared to the parental genotypes M82 and S. pennellii over three consecutive trials. Comparative microarray analysis of IL 12-4 and M82 fruits over a 2-year period allowed 253 differentially-expressed genes to be identified, suggesting that AsA accumulation in IL 12-4 may be caused by a combination of increased metabolic flux and reduced utilization of AsA. In particular, the upregulation of a pectinesterase and two polygalacturonases suggests that AsA accumulation in IL12-4 fruit is mainly achieved by increasing flux through the L-galactonic acid pathway, which is driven by pectin degradation and may be triggered by ethylene.ConclusionsBased on functional annotation, gene ontology classification and hierarchical clustering, a subset of the 253 differentially-expressed transcripts was used to develop a model to explain the higher AsA content in IL 12-4 fruits in terms of metabolic flux, precursor availability, demand for antioxidants, abundance of reactive oxygen species and ethylene signaling.

An association mapping approach to identify favourable alleles for tomato fruit quality breeding

BackgroundGenome Wide Association Studies (GWAS) have been recently used to dissect complex quantitative traits and identify candidate genes affecting phenotype variation of polygenic traits. In order to map loci controlling variation in tomato marketable and nutritional fruit traits, we used a collection of 96 cultivated genotypes, including Italian, Latin American, and other worldwide-spread landraces and varieties. Phenotyping was carried out by measuring ten quality traits and metabolites in red ripe fruits. In parallel, genotyping was carried out by using the Illumina Infinium SolCAP array, which allows data to be collected from 7,720 single nucleotide polymorphism (SNP) markers.ResultsThe Mixed Linear Model used to detect associations between markers and traits allowed population structure and relatedness to be evidenced within our collection, which have been taken into consideration for association analysis. GWAS identified 20 SNPs that were significantly associated with seven out of ten traits considered. In particular, our analysis revealed two markers associated with phenolic compounds, three with ascorbic acid, β-carotene and trans-lycopene, six with titratable acidity, and only one with pH and fresh weight. Co-localization of a group of associated loci with candidate genes/QTLs previously reported in other studies validated the approach. Moreover, 19 putative genes in linkage disequilibrium with markers were found. These genes might be involved in the biosynthetic pathways of the traits analyzed or might be implied in their transcriptional regulation. Finally, favourable allelic combinations between associated loci were identified that could be pyramided to obtain new improved genotypes.ConclusionsOur results led to the identification of promising candidate loci controlling fruit quality that, in the future, might be transferred into tomato genotypes by Marker Assisted Selection or genetic engineering, and highlighted that intraspecific variability might be still exploited for enhancing tomato fruit quality.

Identification of candidate genes for phenolics accumulation in tomato fruit

Phenolics are antioxidants present in tomato fruit that confer healthy benefits and exhibit crucial roles for plant metabolism and response to environmental stimuli. An approach based on two genomics platforms was undertaken to identify candidate genes associated to higher phenolics content in tomato fruit. A comparative transcriptomic analysis between the S. pennellii Introgression Line 7-3, which produced an average higher level of fruit phenolics, and the cultivated variety M82, revealed that their differences are attributed to genes involved in phenolics accumulation into the vacuole. The up-regulation of genes coding for one MATE-transporter, one vacuolar sorting protein and three GSTs supported this hypothesis. The observed balancing effect between two ethylene responsive factors (ERF1 and ERF4) was also hypothesized to drive the transcriptional regulation of these transport genes. In order to confirm such model a TILLING platform was explored. A mutant was isolated harbouring a point mutation in the ERF1 cds that affects the protein sequence and its expected function. Fruits of the mutant exhibited a significant reduced level of phenolics than the control variety. Changes in the expression of genes involved in sequestration of phenolics in vacuole also supported the hypothesized key-role of ERF1 in orchestrating these genes.

Quantitative trait loci pyramiding for fruit quality traits in tomato

Fruit quality is a major focus for most conventional and innovative tomato breeding strategies, with particular attention being paid to fruit antioxidant compounds. Tomatoes represent a major contribution to dietary nutrition worldwide and a reservoir of diverse antioxidant molecules. In a previous study, we identified two Solanum pennellii introgression lines (IL7-3 and IL12-4) harbouring quantitative trait loci (QTL) that increase the content of ascorbic acid (AsA), phenols and soluble solids (degrees Brix; °Bx) in tomato fruit. The purpose of the present work was to pyramid into cultivated varieties the selected QTL for enhanced antioxidant and °Bx content. To better understand the genetic architecture of each QTL, the two ILs were crossed to the recurrent parent M82 (ILH7-3 and ILH12-4) and between them (ILH7-3+12-4). F1 hybrids (ILH7-3+12-4) were then selfed up to obtain F3 progenies in order to stabilize the favourable traits at the homozygous condition. Species-specific molecular markers were identified for each introgressed region and allowed us to select four F2 genotypes carrying both introgressions at the homozygous condition. The F3 double homozygous plants displayed AsA, total phenols and °Bx content significantly higher than M82. Therefore, they may represent suitable genetic material for breeding schemes aiming to increase antioxidant content in tomato fruit.

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.

Exploring a Tomato Landraces Collection for Fruit-Related Traits by the Aid of a High-Throughput Genomic Platform.

During its evolution and domestication Solanum lycopersicum has undergone various genetic ‘bottlenecks’ and extreme inbreeding of limited genotypes. In Europe the tomato found a secondary centre for diversification, which resulted in a wide array of fruit shape variation given rise to a range of landraces that have been cultivated for centuries. Landraces represent a reservoir of genetic diversity especially for traits such as abiotic stress resistance and high fruit quality. Information about the variation present among tomato landrace populations is still limited. A collection of 123 genotypes from different geographical areas was established with the aim of capturing a wide diversity. Eighteen morphological traits were evaluated, mainly related to the fruit. About 45% of morphological variation was attributed to fruit shape, as estimated by the principal component analysis, and the dendrogram of relatedness divided the population in subgroups mainly on the basis of fruit weight and locule number. Genotyping was carried out using the tomato array platform SolCAP able to interrogate 7,720 SNPs. In the whole collection 87.1% markers were polymorphic but they decreased to 44–54% when considering groups of genotypes with different origin. The neighbour-joining tree analysis clustered the 123 genotypes into two main branches. The STRUCTURE analysis with K = 3 also divided the population on the basis of fruit size. A genomic-wide association strategy revealed 36 novel markers associated to the variation of 15 traits. The markers were mapped on the tomato chromosomes together with 98 candidate genes for the traits analyzed. Six regions were evidenced in which candidate genes co-localized with 19 associated SNPs. In addition, 17 associated SNPs were localized in genomic regions lacking candidate genes. The identification of these markers demonstrated that novel variability was captured in our germoplasm collection. They might also provide a viable indirect selection tool in future practical breeding programs.

Unraveling the complexity of transcriptomic, metabolomic and quality environmental response of tomato fruit

BackgroundThe environment has a profound influence on the organoleptic quality of tomato (Solanum lycopersicum) fruit, the extent of which depends on a well-regulated and dynamic interplay among genes, metabolites and sensorial attributes. We used a systems biology approach to elucidate the complex interacting mechanisms regulating the plasticity of sensorial traits. To investigate environmentally challenged transcriptomic and metabolomic remodeling and evaluate the organoleptic consequences of such variations we grown three tomato varieties, Heinz 1706, whose genome was sequenced as reference and two “local” ones, San Marzano and Vesuviano in two different locations of Campania region (Italy).ResultsResponses to environment were more pronounced in the two “local” genotypes, rather than in the Heinz 1706. The overall genetic composition of each genotype, acting in trans, modulated the specific response to environment. Duplicated genes and transcription factors, establishing different number of network connections by gaining or losing links, play a dominant role in shaping organoleptic profile. The fundamental role of cell wall metabolism in tuning all the quality attributes, including the sensorial perception, was also highlighted.ConclusionsAlthough similar fruit-related quality processes are activated in the same environment, different tomato genotypes follow distinct transcriptomic, metabolomic and sensorial trajectories depending on their own genetic makeup.

Exploiting Genomics Resources to Identify Candidate Genes Underlying Antioxidants Content in Tomato Fruit

The tomato is a model species for fleshy fruit development and ripening, as well as for genomics studies of others Solanaceae. Many genetic and genomics resources, including databases for sequencing, transcriptomics and metabolomics data, have been developed and are today available. The purpose of the present work was to uncover new genes and/or alleles that determine ascorbic acid and carotenoids accumulation, by exploiting one Solanum pennellii introgression lines (IL7-3) harboring quantitative trait loci (QTL) that increase the content of these metabolites in the fruit. The higher ascorbic acid and carotenoids content in IL7-3 was confirmed at three fruit developmental stages. The tomato genome reference sequence and the recently released S. pennellii genome sequence were investigated to identify candidate genes (CGs) that might control ascorbic acid and carotenoids accumulation. First of all, a refinement of the wild region borders in the IL7-3 was achieved by analyzing CAPS markers designed in our laboratory. Afterward, six CGs associated to ascorbic acid and one with carotenoids metabolism were identified exploring the annotation and the Gene Ontology terms of genes included in the region. Variants between the sequence of the wild and the cultivated alleles of these genes were investigated for their functional relevance and their potential effects on the protein sequences were predicted. Transcriptional levels of CGs in the introgression region were extracted from RNA-Seq data available for the entire S. pennellii introgression lines collection and verified by Real-Time qPCR. Finally, seven IL7-3 sub-lines were genotyped using 28 species-specific markers and then were evaluated for metabolites content. These analyses evidenced a significant decrease in transcript abundance for one 9-cis-epoxycarotenoid dioxygenase and one L-ascorbate oxidase homolog, whose role in the accumulation of carotenoids and ascorbic acid is discussed. Comprehensively, the reported results demonstrated that combining genetic and genomic resources in tomato, including bioinformatics tools, was a successful strategy to dissect one QTL for the increase of ascorbic acid and carotenoids in tomato fruit.

Comparative transcriptomic profiling of two tomato lines with different ascorbate content in the fruit.

In recent years, interest in tomato breeding for enhanced antioxidant content has increased as medical research has pointed to human health benefits from antioxidant dietary intake. Ascorbate is one of the major antioxidants present in tomato, and little is known about mechanisms governing ascorbate pool size in this fruit. In order to provide further insights into genetic mechanisms controlling ascorbate biosynthesis and accumulation in tomato, we investigated the fruit transcriptome profile of the Solanum pennellii introgression line 10-1 that exhibits a lower fruit ascorbate level than its cultivated parental genotype. Our results showed that this reduced ascorbate level is associated with an increased antioxidant demand arising from an accelerated oxidative metabolism mainly involving mitochondria, peroxisomes, and cytoplasm. Candidate genes for controlling ascorbate level in tomato fruit were identified, highlighting the role of glycolysis, glyoxylate metabolism, and purine breakdown in modulating the ascorbate pool size.

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

Tomato is one of the most important vegetable crop worldwide with a total production of around 141 million tons on a cultivated area of around 5 million hectares (FAOSTAT, 2009, http://faostato.fao.org). Among the most representative countries, Italy contributes with more than 6 million tons to the world production, on a cultivated area of around 117.000 hectares, both in open fields and greenhouses (FAOSTAT, 2009). This crop represents also one of the major products of the food industry worldwide and Italy ranks first for processing tomato production among Countries of the Mediterranean Region (World Processing Tomato Council, 2009, www.wptc.to). Indeed, the high variability of tomato fruits, ranging from the cherry type to the big round or elongated berry, supplies both fresh market and processing products, such as paste, juice, sauce, powder or whole. In the last years, tomato consumption has further increased since it was demonstrated that tomato fruit could protect against diseases, such as cancer and cardiovascular disorders, due to its antioxidant properties (Rein et al., 2006). Tomato fruits are particularly rich of nutritional compounds such as lycopene and alfa-carotene, vitamin C, flavonoids and hydroxycinnamic acid derivatives whose intake would account for health benefits. The cultivated tomato (Solanum lycopersicum) belongs to the Solanaceae family that includes more than 3.000 species, among which 12 represent tomato wild relatives. These species exhibit a wide variety of adaptation to diverse habitats, plant morphology, fruit size and colour, the latter varying from green to white, yellow, pink, red, brown, depending mainly on the metabolites fruit content. The wild related tomato species represent a potential reservoir of useful genes that have been greatly used in breeding programs (Bai & Lindhout, 2007; Gur & Zamir, 2004). Indeed, this vegetable is one of the most investigated crop both at genetic and genomic level not only because of its economic importance but also because it is one of the best characterized plant systems. It has diploid genetics (24 somatic chromosomes), a small genome size (950 Mb per haploid nucleus), is self-pollinated, has a short generation time, is easily reproduced by seed and vegetative propagation and is crosscompatible with many wild species. All these characteristics make it amenable to genetic analysis.