Maria Raffaella Ercolano

Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq

BackgroundThe availability of draft crop plant genomes allows the prediction of the full complement of genes that encode NB-LRR resistance gene homologs, enabling a more targeted breeding for disease resistance. Recently, we developed the RenSeq method to reannotate the full NB-LRR gene complement in potato and to identify novel sequences that were not picked up by the automated gene prediction software. Here, we established RenSeq on the reference genome of tomato (Solanum lycopersicum) Heinz 1706, using 260 previously identified NB-LRR genes in an updated Solanaceae RenSeq bait library.ResultUsing 250-bp MiSeq reads after RenSeq on genomic DNA of Heinz 1706, we identified 105 novel NB-LRR sequences. Reannotation included the splitting of gene models, combination of partial genes to a longer sequence and closing of assembly gaps. Within the draft S. pimpinellifolium LA1589 genome, RenSeq enabled the annotation of 355 NB-LRR genes. The majority of these are however fragmented, with 5′- and 3′-end located on the edges of separate contigs. Phylogenetic analyses show a high conservation of all NB-LRR classes between Heinz 1706, LA1589 and the potato clone DM, suggesting that all sub-families were already present in the last common ancestor. A phylogenetic comparison to the Arabidopsis thaliana NB-LRR complement verifies the high conservation of the more ancient CCRPW8-type NB-LRRs. Use of RenSeq on cDNA from uninfected and late blight-infected tomato leaves allows the avoidance of sequence analysis of non-expressed paralogues.ConclusionRenSeq is a promising method to facilitate analysis of plant resistance gene complements. The reannotated tomato NB-LRR complements, phylogenetic relationships and chromosomal locations provided in this paper will provide breeders and scientists with a useful tool to identify novel disease resistance traits. cDNA RenSeq enables for the first time next-gen sequencing approaches targeted to this very low-expressed gene family without the need for normalization.

PRGdb 2.0: towards a community-based database model for the analysis of R-genes in plants

The Plant Resistance Genes database (PRGdb; http://prgdb.org) is a comprehensive resource on resistance genes (R-genes), a major class of genes in plant genomes that convey disease resistance against pathogens. Initiated in 2009, the database has grown more than 6-fold to recently include annotation derived from recent plant genome sequencing projects. Release 2.0 currently hosts useful biological information on a set of 112 known and 104 310 putative R-genes present in 233 plant species and conferring resistance to 122 different pathogens. Moreover, the website has been completely redesigned with the implementation of Semantic MediaWiki technologies, which makes our repository freely accessed and easily edited by any scientists. To this purpose, we encourage plant biologist experts to join our annotation effort and share their knowledge on resistance-gene biology with the rest of the scientific community.

Overview of tomato (Solanum lycopersicum) candidate pathogen recognition genes reveals important Solanum R locus dynamics

To investigate the genome-wide spatial arrangement of R loci, a complete catalogue of tomato (Solanum lycopersicum) and potato (Solanum tuberosum) nucleotide-binding site (NBS) NBS, receptor-like protein (RLP) and receptor-like kinase (RLK) gene repertories was generated. Candidate pathogen recognition genes were characterized with respect to structural diversity, phylogenetic relationships and chromosomal distribution. NBS genes frequently occur in clusters of related gene copies that also include RLP or RLK genes. This scenario is compatible with the existence of selective pressures optimizing coordinated transcription. A number of duplication events associated with lineage-specific evolution were discovered. These findings suggest that different evolutionary mechanisms shaped pathogen recognition gene cluster architecture to expand and to modulate the defence repertoire. Analysis of pathogen recognition gene clusters associated with documented resistance function allowed the identification of adaptive divergence events and the reconstruction of the evolution history of these loci. Differences in candidate pathogen recognition gene number and organization were found between tomato and potato. Most candidate pathogen recognition gene orthologues were distributed at less than perfectly matching positions, suggesting an ongoing lineage-specific rearrangement. Indeed, a local expansion of Toll/Interleukin-1 receptor (TIR)-NBS-leucine-rich repeat (LRR) (TNL) genes in the potato genome was evident. Taken together, these findings have implications for improved understanding of the mechanisms of molecular adaptive selection at Solanum R loci.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives

The draft genome and transcriptome sequences of the wild potato species S. commersonii demonstrate the usefulness of genome sequences from wild relatives for elucidating evolutionary mechanisms contributing to Solanum species diversity and understanding changes in response to cold. Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.

Genome-wide analysis of histone modifiers in tomato: gaining an insight into their developmental roles

BackgroundHistone post-translational modifications (HPTMs) including acetylation and methylation have been recognized as playing a crucial role in epigenetic regulation of plant growth and development. Although Solanum lycopersicum is a dicot model plant as well as an important crop, systematic analysis and expression profiling of histone modifier genes (HMs) in tomato are sketchy.ResultsBased on recently released tomato whole-genome sequences, we identified in silico 32 histone acetyltransferases (HATs), 15 histone deacetylases (HDACs), 52 histone methytransferases (HMTs) and 26 histone demethylases (HDMs), and compared them with those detected in Arabidopsis (Arabidopsis thaliana), maize (Zea mays) and rice (Oryza sativa) orthologs. Comprehensive analysis of the protein domain architecture and phylogeny revealed the presence of non-canonical motifs and new domain combinations, thereby suggesting for HATs the existence of a new family in plants. Due to species-specific diversification during evolutionary history tomato has fewer HMs than Arabidopsis. The transcription profiles of HMs within tomato organs revealed a broad functional role for some HMs and a more specific activity for others, suggesting key HM regulators in tomato development. Finally, we explored S. pennellii introgression lines (ILs) and integrated the map position of HMs, their expression profiles and the phenotype of ILs. We thereby proved that the strategy was useful to identify HM candidates involved in carotenoid biosynthesis in tomato fruits.ConclusionsIn this study, we reveal the structure, phylogeny and spatial expression of members belonging to the classical families of HMs in tomato. We provide a framework for gene discovery and functional investigation of HMs in other Solanaceae species.

Structural and Functional Genomics of Tomato

Tomato (Solanum lycopersicum L.) is the most intensively investigated Solanaceous species both in genetic and genomics studies. It is a diploid species with a haploid set of 12 chromosomes and a small genome (950 Mb). Based on the detailed knowledge on tomato structural genomics, the sequencing of the euchromatic regions started in the year 2005 as a common effort of different countries. The manuscript focuses on markers used for tomato, on mapping efforts mainly based on exploitation of natural biodiversity, and it gives an updated report on the international sequencing activities. The principal tools developed to explore the function of tomato genes are also summarized, including mutagenesis, genetic transformation, and transcriptome analysis. The current progress in bioinformatic strategies available to manage the overwhelming amount of data generated from different tomato “omics” approaches is reported, and emphasis is given to the effort of producing a computational workbench for the analysis of the organization, as well as the functionality and evolution of the Solanaceae family.

Use of network analysis to capture key traits affecting tomato organoleptic quality

The long-term objective of tomato breeders is to identify metabolites that contribute to defining the target flavour and to design strategies to enhance it. This paper reports the results of network analysis, based on metabolic phenotypic and sensory data, to highlight important relationships among such traits. This tool allowed a reduction in data set complexity, building a network consisting of 35 nodes and 74 links corresponding to the 74 significant (positive or negative) correlations among the variables studied. A number of links among traits contributing to fruit organoleptic quality and to the perception of sensory attributes were identified. Modular partitioning of the characteristics involved in fruit organoleptic perception captured the essential fruit parameters that regulate interactions among different class traits. The main feature of the network was the presence of three nodes interconnected among themselves (dry matter, pH, and °Brix) and with other traits, and nodes with widely different linkage degrees. Identification of strong associations between some metabolic and sensory traits, such as citric acid with tomato smell, glycine with tomato smell, and granulosity with dry matter, suggests a basis for more targeted investigations in the future.

Genetic diversity of Spanish Cucurbita pepo landraces: an unexploited resource for summer squash breeding

Cucurbita pepo is a worldwide cultivated vegetable of American origin. Most of the widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). These forms were developed in Europe after the arrival of the first American landraces through a process of selection and fixation that led to a loss of genetic diversity. Part of the genetic variability of the first American cultigens remains intact in diverse landraces that are still cultivated for self-consumption and sale in local markets. Using the first collection of genomic and EST-derived microsatellites that has just become available for the species, we compared the natural variation present in a collection of Spanish landraces with that of a set of commercial varieties and hybrids, representing current summer squash market offerings. A total of 194 alleles allowed us to distinguish all the genotypes, even those that were closely related. In general, Cocozelle and Vegetable marrow, groups with considerably long histories, were more variable than the Zucchini group, of more recent origin. We found significant genetic diversity among landraces. The variation present among landraces belonging to the Zucchini group was larger than that of the commercial cultivars. Cluster, principal coordinate and population structure results suggested that the variation of the Spanish landraces has not been extensively used in breeding. Commercial summer squashes can therefore benefit from this underexploited variability, especially from certain landraces that already display favourable commercial traits.

Genetic and genomic approaches for R-gene mediated disease resistance in tomato: retrospects and prospects.

Tomato (Solanum lycopersicum) is one of the world’s most important vegetable crops. Managing the health of this crop can be particularly challenging; crop resistance may be overcome by new pathogen races while new pathogens have been introduced by global agricultural markets. Tomato is extensively used as a model plant for resistance studies and much has been attained through both genetic and biotechnological approaches. In this paper, we illustrate genomic methods currently employed to preserve resistant germplasm and to facilitate the study and transfer of resistance genes, and we describe the genomic organization of R-genes. Patterns of gene activation during disease resistance response, identified through functional approaches, are depicted. We also describe the opportunities offered by the use of new genomic technologies, including high-throughput DNA sequencing, large-scale expression data production and the comparative hybridization technique, whilst reporting multifaceted approaches to achieve genetic tomato disease control. Future strategies combining the huge amount of genomic and genetic data will be able to accelerate development of novel resistance varieties sustainably on a worldwide basis. Such strategies are discussed in the context of the latest insights obtained in this field.

Evaluation and use of plant biodiversity for food and pharmaceuticals.

Many epidemiological studies have shown the importance of fruit and vegetables in the human diet so as to prevent the onset of cardiovascular disease and several forms of cancer. The use for food and pharmaceuticals of two of the most widely grown and genetically well-known species in the world, the tomato and the potato, is reviewed. Tomatoes are important sources of vitamin C, potassium, folic acid and carotenoids such as lycopene and beta-carotene. It has been demonstrated that lycopene has anti-oxidant properties and interferes with the growth of cancerous cells. At the Department of Agronomy and Plant Genetics in Portici, interesting results have been obtained with the constitution of stable tomato hybrids having a high content of lycopene and vitamin C. Many of the parental lines used in constituting the hybrids come from interspecific crosses. Potato is also very important in the human diet for its content of high quality proteins, mineral salts and vitamins and it has many medicinal properties. The use of diploid wild species to transfer traits such as high content of vitamin C, mineral salts and high quality proteins into the cultivated potato through ploidy manipulation is discussed.