The distribution of telomeric repeats was analyzed by fluorescence in situ hybridization in 15 species of arvicoline rodents, included in three different genera: Chionomys, Arvicola, and Microtus. The results demonstrated that in most or the analyzed species, telomeric sequences are present, in addition to normal telomeres localization, as large blocks in pericentromeric regions. The number, localization, and degree of amplification of telomeric sequences blocks varied with the karyotype and the morphology of the chromosomes. Also, in some cases telomeric amplification at non-pericentromeric regions is described. The interstitial telomeric sequences are evolutionary modern and have rapidly colonized and spread in pericentromeric regions of chromosomes by different mechanisms and probably independently in each species. Additionally, we colocalized telomeric repeats and the satellite DNA Msat-160 (also located in pericentromeric regions) in three species and cloned telomeric repeats in one of them. Finally, we discuss about the possible origin and implication of telomeric repeats in the high rate of karyotypic evolution reported for this rodent group.

In the subfamily Arvicolinae (Cricetidae, Rodentia) the satellite DNA Msat-160 has been so far described in only some species from the genus Microtus and in one species from another genus, Chionomys nivalis. Here we cloned and characterized this satellite in two new arvicoline species, Microtus (Terricola) savii and Arvicola amphibius (terrestris). We have also demonstrated, by PCR and FISH, its existence in the genomes of several other species from both genera. These results suggest that Msat-160 already occurred in the common ancestor of the four genera/subgenera of Arvicolinae (Microtus, Chionomys, Arvicola, and Terricola). In Arvicola and Terricola, Msat-160 showed the basic monomer length of 160 bp, although a higher-order repeat (HORs) of 640 bp could have been probably replacing the original monomeric unit in A. a. terrestris. Msat-160 was localized by FISH mostly on the pericentromeric regions of the chromosomes, but the signal intensity and the number of carrier chromosomes varied extremely even between closely related species, resulting in a species-specific pattern of chromosomal distribution of this satellite. Such a variable pattern most likely is a consequence of a rapid amplification and contraction of particular repeats in the pericentromeric regions of chromosomes. In addition, we proposed that the rapid variation of pericentromeric repeats is strictly related to the prolific species radiation and diversification of karyotypes that characterize Arvicolinae lineage. Finally, we performed phylogenetic analysis in this group of related species based on Msat-160 that results to be in agreement with previously reported phylogenies, derived from other molecular markers.

Sex chromosome evolution in mammals has been extensively investigated through chromosome-painting analyses. In some rodent species from the subfamily Arvicolinae the sex chromosomes contain remarkable features such as giant size, a consequence of heterochromatic enlargement, or asynaptic behaviour during male meiosis. Here, we have made a comparative study of the sex chromosomes in 6 arvicolid species using different probes from the X and Y chromosomes of 3 species, in order to gain knowledge about intra- or interspecific preservation of euchromatic regions. Our results clearly reveal poor conservation of the euchromatic region of the Y chromosome within these species, while the euchromatin on the X chromosome is extremely well preserved. Furthermore, we detected no clear correlation between the synaptic/asynaptic behaviour of the sex chromosomes, and the presence or absence of sequence homology within their euchromatic regions. Notably, our study has shown a new relationship between the giant sex chromosomes of 2 species, Microtus agrestis and Microtus cabrerae, that is, both X and Y share a novel region of common sequences in the euchromatin that is not present in the other species analysed. This interspecific euchromatic conservation, limited to the giant sex chromosomes, could point towards a common evolutionary origin for the heterochromatic enlargement process that has characterized the evolution of the sex chromosomes in some arvicolid species.

A new repeated DNA from Microtus thomasi, Mth-Alu2.2, was cloned and characterized and is presented here for the first time. Digestion of genomic DNA from M. thomasi with AluI restriction enzyme revealed a 2.2-kb repetitive DNA sequence with a high AT content (69%). This sequence consists of a tandemly repeated nonanucleotide of the consensus sequence CACAATGTA, which constitutes approximately 93-95% of the total unit length. The location of the Mth-Alu2.2 sequence in the karyotype was determined by FISH, demonstrating strong hybridization signals in the pericentromeric regions of all chromosomes and in the heterochromatin blocks of several X chromosome variants. In addition, the distribution of the 4 pericentromeric repeat sequences Msat-160, Mth-Alu900, Mth-Alu2.2, and interstitial telomeric repeats was analyzed by in situ hybridization in M. thomasi, in order to shed light on the complex composition of the chromosomal pericentromeric regions in this species. The order and organization of these sequences in the pericentromeric regions are conserved, with slight variations in both the degree of overlapping and the amount of each repeated DNA in the chromosomes. Specifically, Mth-Alu2.2 is localized in the terminal regions of the chromosomes, with Msat-160 occupying the immediately inner region, partially intermixed with Mth-Alu2.2. The sequence Mth-Alu900 is found in internal positions below Msat-160, and the interstitial telomeric repeats are located close to the long-arm euchromatin of the chromosomes.

The sibling species Microtus thomasi and M. atticus represent probably the highest karyotypic diversity within the genus Microtus and are an interesting model for chromosomal evolution studies. In addition to variation in autosomes, they show a high intraspecific variation in the size and morphology of both sex chromosomes. We analyzed individuals with different sex chromosome constitutions using 3 painting probes, 2 from Y chromosome variants and 1 from the small arm of the submetacentric X chromosome. Our comparative painting approach uncovered 12 variants of Y and 14 variants of X chromosomes, which demonstrates that the polymorphism of sex chromosomes is substantially larger than previously reported. We suggest that 2 main processes are responsible for this sex chromosome polymorphism: change of morphology from acrocentric to submetacentric or metacentric chromosomes and increase in size due to accumulation of repetitive DNA sequences, generating heterochromatic blocks. Strong genetic drift in small and fragmented populations of these 2 species could be related to the origin and maintenance of the large polymorphism of sex chromosomes. We proposed that a similar polymorphism variation combined with random drift fixing the biggest sex chromosomes could have occurred in the origin of some of the actual Microtus species with giant sex chromosomes.