In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years). These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.

The concentration of evolutionary breakpoints in primate karyotypes in some particular regions or chromosome bands suggests that these chromosome regions are more prone to breakage. This is the first extensive comparative study which investigates a possible relationship of two genetic markers (intrachromosomal telomeric sequences [TTAGGG]n, [ITSs] and fragile sites [FSs]), which are implicated in the evolutionary process as well as in chromosome rearrangements. For this purpose, we have analyzed: (a) the cytogenetic expression of aphidicolin-induced FSs in Cebus apella and Cebus nigrivittatus (F. Cebidae, Platyrrhini) and Mandrillus sphinx (F. Cercopithecidae, Catarrhini), and (b) the intrachromosomal position of telomeric-like sequences by FISH with a synthetic (TTAGGG)n probe in C. apella chromosomes. The multinomial FSM statistical model allowed us to determinate 53 FSs in C. apella, 16 FSs in C. nigrivittatus and 50 FSs in M. sphinx. As expected, all telomeres hybridized with the probe, and 55 intrachromosomal loci were also detected in the Cebus apella karyotype. The χ² test indicates that the coincidence of the location of Cebus and Mandrillus FSs with the location of human FSs is significant (P < 0.005). Based on a comparative cytogenetic study among different primate species we have identified (or described) the chromosome bands in the karyotypes of Papionini and Cebus species implicated in evolutionary reorganizations. More than 80% of these evolutionary breakpoints are located in chromosome bands that express FSs and/or contain ITSs.

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