In Silico Detection and FISH Analysis to Determine Location of miRNAs in Solea Senegalensis Chromosomes Using BACs

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play a very important role in gene expression through regulation of mRNA cleavage and translation. The Senegalese sole, Solea senegalensis (Kaup 1858), is a flatfish species that shows great potential for marine aquaculture. Nevertheless, the existence of sexual dysfunction of males reared in captivity, high larval mortality and diseases have hampered its production. The integration of sequence information with data on chromosomal physical location is useful for understanding genetic processes and is essential for comparative analysis, QTL mapping and positional cloning of genes. In this study we employed an in silico approach to identify potential miRNAs in a S. senegalensis BAC library. Mature sequences were obtained from miRBase and other sources and a BLAST analysis was carried out against sequences from a S. senegalensis BAC library. Sequences showing similarity were extracted and subjected to an analysis of their secondary structure. This information was combined with BAC-FISH data to assign miRNAs to chromosomes. In addition, a phylogenetic analysis of some miRNAs families was carried out and target genes were predicted against a reference transcriptome. After secondary structure analysis, 45 groups of sequences showed similarity to mature miRNAs. Of these, 19 were assigned to Senegalese Sole chromosomes using BAC-FISH data and thirty-four unique mature sequences targeted 12387 sequences from a S. senegalensis reference transcriptome. Phylogenetic trees showed different levels of support for clustering among types of miRNAs and S. senegalensis sequences usually were closed to other Pleuronectiformes. These results will be helpful to understand how miRNAs affect gene expression in this species and biological processes of interest for its aquaculture. Nevertheless, experimental validation through, for example, RT-PCR, northern blotting or microarrays is required.