Genetic evolution of the pathway controlling fruit carotenoid content in tomato (S. lycopersicum) and its wild relatives

Tomato (Solanum lycopersicum) is an economically and nutritionally valuable crop and constitutes a model plant for genetic research of the Solanaceae family thanks its compact genome (950 Mb), short generation time, the availability of a large set of mutants, routine transformation technology and availability of rich genetic and genomic resources. The pre-release of the tomato genome sequence, produced combining a whole genome shotgun and a BAC-by-BAC approach, has been announced at the end of 2009 and a first genome annotation is available and is continuously updated. The genome of cultivated tomato, however, has a limited sequence variation due to bottlenecks during domestication and breeding. The wild tomatoes are native to Western South America and grow in a variety of habitats, from near sea level along the arid Pacific coast to high mountains up to 3300 m. Thanks to their evolutionary and adaptive history, the wild tomato species contain useful traits that can be introgressed into cultivated tomato, such as tolerance to drought and salinity and resistance to multiple pathogens. In recent years there has been an increasing interest in analyzing various biological properties of natural genetic diversity and wild species provide a wealth of useful genetic traits to improve cultivated tomatoes. The evolution of carotenoid pigments affecting berry colour is one of the variable characters within tomato and its wild relatives that have obtained much attention: the fruit colour of wild tomatoes varies from green to orange and red. Numerous studies have demonstrated that green fruit is the ancestral character. The reason behind a transition to coloured fruits during the evolution of the group is still not clear, but probably is related to attraction of animals for seed dispersal. Accumulation of carotenoids as secondary metabolites in fruits and flowers occurs through up-regulation of the pathway at the gene expression level. A candidate gene approach has been used, focusing our attention on the carotenoid biosynthetic pathway. Several genes of this pathway have been sequenced in the wild tomato species with different berry colour. Accessions of the S. lycopersicum var. cerasiforme as well as wild tomato species (S. pimpinellifolium, S. cheesmaniae, S. neorickii, S. chmielewskii, S. chilense, S. habrochaites, S. pennellii, S. arcanum) were selected to represent ancestral and closely related progenitor genotypes. Thanks to the availability of the genome sequences and annotation data of tomato, potato and A. thaliana, a comparison was undertaken to investigate the presence/absence of microsynteny around the Phytoene Synthase genes among these organisms. Moreover, we have obtained structural information on the organization of the carotenoid genes object of this thesis. The analysis has highlighted the presence of three new genes, annotated respectively as putative LCY-b, putative CrtISO and putative LCY-b: from preliminary transcriptome data the genes codifying for these proteins seem to be expressed. The sequencing of carotenoid genes from PSY down to LCY-e (-branch) and CHY1-2 (-branch) has been completed for all the wild species studied in this thesis. The sequence analysis has highlighted the presence of numerous mutations. Some non-synonymous substitutions are candidate to be hypo- or hypermorphic alleles. Preliminary analysis on in vitro protein expression seems to confirm that one of the green-fruited species analysed carries a hypermorphic allele of LCY-e. Green-fruited species did not differ systematically from coloured-fruited ones in climacteric ethylene production or fruit softening, with the exception of S. arcanum, which shows an ethylene peak just before fruit abscission. Carotenoid and gene expression profiles of the red-fruited species, S. lycopersicum and S. pimpinellifolium, are very similar, in agreement with their phylogenetic closeness. In contrast to expectations, orange-fruited S. cheesmaniae has the lowest carotenoid content among all species and has carotenoid and transcriptional profiles similar to the green-fruited species.