Análisis estructural comparativo de los genes de las enterotoxinas de " Escherichia coli "

Resumen

Turbot is one of the most important farmed fish in Europe. A significant drawback for its production is the size dispersion. This species shows extreme growth rate dimorphism between sexes. Males grow slower and achieve sexual maturity earlier than females. For these reasons exists a huge interest of the companies in producing all-female populations. Thus, understanding sex determination (SD) and differentiation mechanisms is a relevant goal for turbot production. Previous studies pointed toward a ZZ/ZW system operating in turbot with influence of minor genetic factors and temperature. Recently, a sex-related QTL analysis based on the genetic turbot map identified a major SD region at linkage group 5 (LG5) and other three suggestive QTL (Quantitative trait loci) in LG6, LG8 and LG21. This approach allowed the detection of a microsatellite marker (SmaUSC-E30) with a high sexing efficiency, which was used to develop a molecular tool to assess the genotypic sex but still presenting important limitations. At this point further research about the sex determination and differentiation was necessary, so during this thesis several analyses were carried out in order to broaden our knowledge on this issue. This work begins with the mapping of several sex-related genes (sox3, sox6, sox8, sox9, sox14, sox17, sox19, amh, cyp19a1a, cyp19a1b and dmrta2) at the turbot linkage map. Interestingly, cyp19a1b mapped close to SD-QTL at LG6 whereas sox9 and sox17 inside that at LG21. Amh and dmrta2, previously suggested as potential master SD key regulators in turbot, were mapped at LG5 but outside the SD-QTL, so they can be likely discarded for this role. On the other hand, in order to identify the SD gene (SDg) a fine mapping was performed at the main SD region. Six genes were selected from a comparative genomics approach, including two strong candidates (sox2 and dnajc19), and were mapped on that region. However, none of them showed association with sex in a wild populations, being also discarded as SDg. The markers located at the SD region were used for a segregation analysis which confirmed a ZZ/ZW mechanism. Furthermore, no recombination suppression or differentiation between males and females was detected at this region, supporting a recent origin of the sex chromosome pair of turbot. All the markers developed were also used for the screening of a BAC (Bacterial artificial chromosome) library. The clones isolated were hybridized on metaphase preparations, revealing LG5 as a medium size acrocentric chromosome and allowing the integration of the cytogenetic and genetic map, which was consolidated from 24 LGs to 22 LGs in accordance with the haploid chromosome composition of this species. Finally, the temporal and spatial expression pattern of several previously mapped genes was analyzed by in situ hybridization with riboprobes on gonad tissue at different stages. During this study, foxl2, amh, sox2, sox3 and sox17 were revealed to be expressed in both males and females. In the case of cyp19a1a and sox19, their expression was restricted to females. The results presented on this thesis enabled a better understanding on the origin and genetic architecture of sex determination and differentiation in turbot, as well as to increase genomic resources in this species useful for future research.