Gene expression study in the non-model organism Botryllus schlosseri through SOLiD RNA-seqs

Botryllus schlosseri is a colonial ascidian widespread in temperate, shallow seas of the world. The organism is widely used for the study, in an evolutionary perspective, of a variety of biological processes ranging from sexual and asexual reproduction to regeneration, allorecognition and immune responses. However, despite its importance as model organism, no sequenced genome is available today. We undertook the analysis of the transcriptome of B. schlosseri in various colonial developmental phases and during tunic regeneration. The asexual reproduction by continuous palleal budding and the vascular system regeneration were the target of our RNA-seq experiments. In the first experiment, 3 different phases of the colonial blastogenetic cycle were considered: the mid-cycle, where colonies are metabolically very active; a phase immediately before the generation change (take-over), where colonies are getting ready to the generation change, and the take-over phase where adult zooids die and are replaced by buds which reach adulthood. Total RNA was extracted from various colonies for each of considered experimental condition. In the second experiment, the tunic of some colonies was cut and let to regenerate for 2 days. After tunic regeneration, total RNA was extracted. cDNA libraries were built according to SOLiD protocols and they were sequenced using SOLiD 4 and SOLID 5500 sequencers. In the absence of a reference genome, the gene expression analysis requires a de novo assembly of RNA-seq experiments. In this thesis a method to assemble RNAseq reads produced by SOLiD sequencers is described for the first time. The analysis of simulated data allowed us to improves the overall method. Gene expression data and gene annotation have been stored in a database and they can be managed in a compact structure which is directly and quickly accessed by a developed Web interface. The Web interface makes possible the analysis of many experimental conditions and their comparison, to highlight expression differences, through a common Web browser. Many thousands of differentially expressed transcripts were found and some of these are involved in natural apoptosis. This biological process was described in details using morphological studies: during take-over, tissues of adult zooids undergo apoptosis and zooids are replaced by primary buds that grow to become the new adult generation.