Elisa Corteggiani Carpinelli

University of Padova | UNIPD · Interdepartmental Research Centre for Innovative Biotechnologies CRIBI

Alternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes. We used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event. As described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures.

Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae.Here we report a high quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of chromosomes. We identified 10,646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing and cell wall synthesis.We also analysed the modification of the transcriptional profile in nitrogen deficiency, a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time we observed a very significant down regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway.Finally we released an information resource of the genomic data of N. gaditana, available at www.nannochloropsis.org.

Next generation sequencing technology has considerably improved over the past few years, making easier and more affordable the shotgun sequencing approach. Short reads are particularly popular as they are very easy and cheap to produce. On the other hand, their assembly results in the generation of a vast number of relatively short contigs that would require suitable physical maps and scaffolding procedures to be further assembled in a draft genomic sequence. Unfortunately, physical maps are still very difficult to produce and take little advantage of the next generation sequencing technology. The aim of our project is to investigate the possibility to overcome this problem. The organism we chose as field test is Nannochloropsis gaditana a unicellular algae that is very promising for biofuel production and has a relatively small genome (32Mb). A whole genome shotgun sequencing project was previously conducted in our laboratory in order to produce a first assembly of the genome. Two runs were performed with Roche 454 GS FLX system.The method we propose is inspired on both Radiation Hybrid 1 and Happy Mapping 2 techniques. The starting point is the creation of a 25X or grater BAC library of the genome of interest. BACs are randomly selected to obtain pools, each pool representing about the 50% of the genome. Individual pools are then sequenced to produce short reads. The rationale of our approach is that the more two loci are close in the genome, the more their corresponding sequences should appear in the same set of pools. Profiles of presence and absence for any locus in the entire set of pools could then be produced, and this information should help in the assembly and scaffolding of genomes.