Evolução cromossômica na família Erythrinidae. Mapeamento citogenético de DNAs repetitivos e microdissecção de cromossomos sexuais
Cioffi, Marcelo de Bello
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The fishes from the Erythrinidae family present a wide karyotypic variation with several karyomorphs already identified for some species, including the presence of different sex chromosome systems. In Erythrinus erythrinus a well-differentiated X1X2Y system occurs in karyomorph D, as well as undifferentiated sex chromosomes in other karyomorphs of this species. In Hoplias malabaricus, single and multiple sex chromosome systems, as well as undifferentiated, can also be found. Among these, there is a well-differentiated XY system in karyomorph B, a nascent XY system in karyomorph C and a X1X2Y system in karyomorph D. The purpose of this thesis was to analyze the genomic differentiation occurred between E. erythrinus and H. malabaricus karyomorphs, with a particular focus on the sex chromosomes. To this end, besides the classical chromosomal analysis, repetitive DNA sequences and microdissected sex chromosomes were used as probes for fluorescence in situ hybridization (FISH) procedures. The evolutionary pathways for the sex chromosome systems of H. malabaricus and E. erythrinus karyomorphs were investigated. Allopatric populations of E. erythrinus karyomorphs A and D were analyzed and showed significant differences in relation to their karyotypes, where chromosomal rearrangements and genomic changes stood out as significant events during the evolutionary process. The genomic dispersion of transposable elements Rex3 associated with the 5S ribosomal DNA, as well as the differentiation of a multiple X1X2Y sex chromosome system were prominent events in the differentiation of karyomorph D. The role of repetitive elements in the process of differentiation of sex chromosomes was also analyzed. In this sense, we performed a comparative study of the distribution of twelve microsatellites (mono-, di-and trinucleotide) in the XY and X1X2Y systems, present in karyomorphs B and D of H. malabaricus, respectively. The differential distribution of microsatellites along the chromosomes in both systems were demonstrated, possibly as a direct reflection of their modes of origin, with a significant accumulation of repeats on the X chromosome of the XY system, in contrast to that found in the X1X2Y sex chromosomes system. The processes acting in the differentiation of sex chromosomes are not yet completely understood. However, the accumulation of repetitive DNA sequences may represent an early step in the differentiation of simple sex chromosomes systems (XY and ZW), highlighting the role of these sequences in their differentiation, and contributing to the reduction of recombination between the undifferentiated ancestor sex pair. On the other hand, in multiple systems, the chromosomal rearrangements implicated in the origin of these systems, appear to represent the primary factors responsible for the reduction of recombination without the need for additional genomic modifications. The comparative mapping of different repetitive DNA sequences in meiotic and mitotic chromosomes pointed for an independent differentiation process of the X1X2Y sex chromosomes in E. erythrinus and H. malabaricus. Additionally, sex chromosomes probes, isolated by microdissection, were used in experiments of whole chromosome painting (wcp), proving to be a powerful tool for the study of sex chromosomes evolution. It was shown that the chromosomes of the X1X2Y system have evolved independently in H. malabaricus and E. erythrinus where different autosomes were firstly converted to a slightly different XY sex pair in each species, followed by dinstinct chromosomal rearrangements in the origin of these systems. In turn, the chromosome painting also showed the independent origin for the XY system of H. malabaricus karyomorphs B and C. Again, distinct autosomal pairs present in the ancestor karyomorphs were converted into the XY chromosomes, now present in karyomorphs B and C. It has been well characterized the direct relationship between the XY system of karyomorph C and the origin of the X1X2Y system present in karyomorph D. Thus, even between congeneric species (E. erythrinus and H. malabaricus), as well as between karyomorphs of the same nominal species (H. malabaricus), the sex chromosomes have evolved independently, demonstrating the great plasticity of this evolutionary process in fishes.