Todesco, Sara (2008) Tomato (Solanum lycopersicum) genome: sequencing and analysis of chromosome 12. [Ph.D. thesis]
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The Solanaceae family includes a number of closely related plant species with diverse phenotypes that have been exploited for agronomic, pharmaceutical and ornamental purposes. In 2003 'The International Solanaceae Genome Project' (SOL) launched the initiative to sequence the 220 Mb of euchromatin of the tomato (Solanum lycopersicum) genome as the central part of a wider project aiming to increase our knowledge about diversity and adaptation in crop species (http://www.sgn.cornell.edu/solanaceae-project/). The sequencing proceeds on a BAC-by-BAC basis with the 12 chromosomes divided over several genomic laboratories of ten different countries. As a member of the project, the Italian research team is involved in the sequencing of the euchromatin portions of chromosome 12.
During my PhD project, I had the opportunity to face this challenging project from different points of view including molecular, cytogenetic and bioinformatic analysis.
A large part of my effort was focused in setting up a sequencing pipeline and starting the construction of a minimal subset of BAC clones covering the chromosome 12 euchromatin with minimal overlaps. The progress can be viewed through the development of the TPF and AGP files, available from the SGN repository (http://www.sgn.cornell.edu/).
A key step for the success of the sequencing project is the identification of a reliable minimal tiling path of neighbouring BAC clones. To improve this process, I contributed to the development of a informatics pipeline called PABS (Platform Assisted BAC-by-BAC Sequencing), freely available to the community at our web site (http://tomato.cribi.unipd.it/files/bioinformatics.html) (Todesco S. et al., 2008). PABS has been specifically designed to minimize the negative impact of genomic repeats, considering that a repeat element can connect non-contiguous regions of the genome,
leading to misalignment of BACs and possible "jumps" along the genome. PABS has two main functions: 1) PABS-Select, to choose suitable overlapping clones for the sequencing walk; 2) PABS-Validate to verify whether a BAC under analysis is actually overlapping the preceding BAC.
A BAC-based physical map is a fundamental tool to further assist the sequencing work but also to connect the minimal tiling path of BACs. In my study, I improved the molecular combing technique (Lebofsky R. et al., 2003; Monier K et al., 2001; Allemand JF et al., 1997) for producing multicolour FISH on stretched genomic DNA molecules. This technique allows accurate mapping of BAC clones and precise measurement of physical distances between contigs with a spatial resolution of 1 to 5 kb.
Finally, to explore the data generated by the BAC-by-BAC sequencing I contributed to a preliminary annotation of the tomato BACs sequences. As a result of this analysis, we outlined some features of the gene organization in the tomato genome.
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