Genomic Organization of Microsatellites and LINE-1-like Retrotransposons: Evolutionary Implications for Ctenomys minutus (Rodentia: Ctenomyidae) Cytotypes

Simple Summary In animals, several species contain substantial chromosomal and genomic variation among their populations, but as to what could have driven such diversification is still a puzzle for most cases. Here, we used molecular cytogenetic analysis to expose the main genomic elements involved in the population variation observed in the Neotropical underground rodents of the genus Ctenomys (Rodentia: Ctenomyidae), which harbor the most significant chromosomal variation among mammals (2n = 10 to 2n = 70). These data provide evidence for a correlation between repetitive genomic content and localization of evolutionary breakpoint regions (EBRs) and highlight their direct impact in promoting chromosomal rearrangements. Abstract The Neotropical underground rodents of the genus Ctenomys (Rodentia: Ctenomyidae) comprise about 65 species, which harbor the most significant chromosomal variation among mammals (2n = 10 to 2n = 70). Among them, C. minutus stands out with 45 different cytotypes already identified, among which, seven parental ones, named A to G, are parapatrically distributed in the coastal plains of Southern Brazil. Looking for possible causes that led to such extensive karyotype diversification, we performed chromosomal mapping of different repetitive DNAs, including microsatellites and long interspersed element-1 (LINE-1) retrotransposons in the seven parental cytotypes. Although microsatellites were found mainly in the centromeric and telomeric regions of the chromosomes, different patterns occur for each cytotype, thus revealing specific features. Likewise, the LINE-1-like retrotransposons also showed a differential distribution for each cytotype, which may be linked to stochastic loss of LINE-1 in some populations. Here, microsatellite motifs (A)30, (C)30, (CA)15, (CAC)10, (CAG)10, (CGG)10, (GA)15, and (GAG)10 could be mapped to fusion of chromosomes 20/17, fission and inversion in the short arm of chromosome 2, fusion of chromosomes 23/19, and different combinations of centric and tandem fusions of chromosomes 22/24/16. These data provide evidence for a correlation between repetitive genomic content and localization of evolutionary breakpoints and highlight their direct impact in promoting chromosomal rearrangements.

Cytotypes with the same 2n are described with "a" or "b", due to the rearrangements in the different chromosomes, allowing them to be differentiated. System "b" chromosome 2 is fissioned, giving rise to two chromosomes (corresponding to 2p and 2q) and a pericentromeric inversion in the chromosome corresponds to 2p. sm = submetacentric; a = acrocentric; * denotes inversion. The karyotype data and the rearrangements involved in their differentiation are by Freitas, Freygang et al., and Lopes et al. [29,31,37]. What could have driven the extensive karyotype diversification observed in these populations? Although the mapping of repetitive sequences has been shown to be useful for detecting karyotypic changes during the chromosomal evolution [41,42], such analyses are still scarce among rodents. As an example, the main 96 repetitive DNA family responsible for rolling circle replication in Ctenomys was isolated and characterized [43,44]. The aim was to investigate the distribution of different repetitive DNAs in order to characterize evolutionary breakpoint regions (EBRs) and to highlight their involvement in promoting chromosomal rearrangements among the seven parapatric parental cytotypes A-G. Additionally, chromosomal mapping of eight microsatellite sequence motifs and LINE-1like retroelement was documented among the populations throughout their distribution What could have driven the extensive karyotype diversification observed in these populations? Although the mapping of repetitive sequences has been shown to be useful for detecting karyotypic changes during the chromosomal evolution [41,42], such analyses are still scarce among rodents. As an example, the main 96 repetitive DNA family responsible for rolling circle replication in Ctenomys was isolated and characterized [43,44]. The aim was to investigate the distribution of different repetitive DNAs in order to characterize evolutionary breakpoint regions (EBRs) and to highlight their involvement in promoting chromosomal rearrangements among the seven parapatric parental cytotypes A-G. Additionally, chromosomal mapping of eight microsatellite sequence motifs and LINE-1-like retroelement was documented among the populations throughout their distribution area (coastal plains of Southern Brazil) here for the first time. Our results demonstrated that DNA satellites are tightly associated with mapped-in EBRs and most likely fostered the extensive karyotype diversification observed.

Sample Collection
Thirteen individuals of C. minutus were collected in the states of Santa Catarina (SC) and Rio Grande do Sul (RS) ( Table 2), using Oneida Victor ® n • 0 weft traps. Animals were euthanized following the guidelines of the Animal Care Committee of the American Society of Mammalogists [45]. The experiments were conducted with the approval of the Ethics Committee for the Use of Animals (CEUA) n • 35,828 of the Universidade Federal do Rio Grande do Sul (Porto Alegre-Brazil), and all field procedures had appropriate permissions from Brazil's Environmental Agency (IBAMA, Authorization n • 14690-1).

Chromosomal Preparations
Chromosomal preparations were obtained from short-term fibroblast cultures [46], with modifications. Tissues from the kidney and/or the lung were disaggregated in collagenase type IV, cultured at 37 • C in DMEM-Dulbecco's Modified Eagle's Mediumhigh glucose (GIBCO™, Grand Island, NY, USA), enriched with 20% fetal bovine serum (GIBCO™, BRL), penicillin (100 units/mL), and streptomycin (100 mg/mL). Cells were grown to~80-90% confluent. For further passages, cells were removed by adding Trypsin-EDTA (GIBCO™, USA), and up to five passages were performed. And at each passage, the chromosomes were obtained by standard protocols: cells were incubated for 3 h with colchicine, treated for 8 min in a hypotonic solution (0.075 M KCl), and fixed in a methanol and acetic acid (3:1) solution. To confirm the diploid number and check if there were no chromosomal alterations, approximately 30 metaphases of each individual were analyzed using 5% Giemsa staining in 0.07 M phosphate buffer (pH 6.8).
The LINE-1-like retroelement was identified and amplified by the polymerase chain reaction (PCR) when using C. minutus (Cytotype A-50a) genomic DNA, which was isolated following [48] and with the set of primers as described by Casavant et al. [49]. The amplified product represented part of the TE reverse transcriptase. The amplicons of approximately 740 bp were purified with a GE Healthcare illustra™ GFX PCR DNA and Gel Band Purification Kit (GE Healthcare UK Ltd., Buckinghamshire, UK), following the manufacturer's recommendations, and then sent for sequencing at Macrogen Inc. (Seoul, Korea). BLAST searches were conducted using the sequencing product against GenBank (https://www.ncbi.nlm.nih.gov/ (accessed on 8 June 2020)) to confirm the identity of the element. Additionally, ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html (accessed on 8 June 2020)) tools were utilized to verify the TE and the domains were identified using the Conserved Domain Database (CDD) platform https://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi (accessed on 8 June 2020). The LINE-1-like retrotransposons (have been deposited in GenBank-OP068276) were used as a template for the PCR labeling; the probe for FISH probe was labeled directly by PCR using Biotin-16-dUTP (Jena Bioscience, Jena, Germany).
The slide preparations, LINE-1-like hybridization, and post-hybridization were performed according to Bertocchi et al. [15], with minor modifications. The hybridization was performed overnight at 37 • C in a humid chamber, and the post-hybridization washes were carried out at 37 • C in 50% formamide for 3 min, followed by two washes in 2× SSC for 5 min each, at 37 • C. The signal was detected using streptavidin-Cy3, and the chromosomes were counterstained by Fluoroshield™ with DAPI (Sigma-Aldrich). For both microsatellites and LINE-1-like FISH experiments, at least 30 metaphases per individual were analyzed to confirm the FISH results and we built karyotypes for each cytotype to ensure correct identification of each chromosome. The slides were analyzed using a Zeiss Axiophot epifluorescence microscope (Zeiss Inc. Carl Zeiss, Heidelberg, Germany), coupled with ZEN BLUE software. Figures were organized using Adobe Photoshop CS6.

Results
The chromosomal mapping of the eight distinct microsatellite motifs revealed that distinct cytotype-specific patterns can occur, although they are generally accumulated in the centromeric and telomeric regions of the chromosomes (Table 3, Figures 2A-H, 3, 4 and S1-S7). Only for the (CGG) 10 probe, no signals of hybridization were observed in the sex chromosomes in any of the seven cytotypes; for the other seven microsatellite probes, signals were observed in the centromeric and terminal regions of the sex chromosomes in the seven cytotypes. Table 3. Hybridization of eight microsatellites in seven cytotypes (A-G) of Ctenomys minutus.

Cytotypes
Motif Probes (A) 30 (C) 30 (CA) 15 (CAC) 10 (CAG) 10 (CGG) 10 (GA) 15 (GAG) 10 Cyt A  The (CA)15 microsatellite shows hybridization signals spread over the entire length of the chromosomes, mainly in the largest ones ( Figure 2B,C). However, the signals varied according to cytotypes. Cytotypes from the north of the geographic distribution present a different distribution when compared to cytotypes from the southern distribution. This difference can be seen, for example, when the distribution of (CA)15 in the cytotype A (50a) is compared with the distribution in the cytotype G (50b). Cytotype A visibly presents more and larger (CA)15 blocks, in contrast to cytotype G (Table 3, Figure 2B and Figure 2C, respectively, and Figures S1-S7).
On the other hand, the microsatellites (C)30, (GAG)10, (CAG)10, and (CAC)10 show a spread distribution, with a preferential accumulation in the telomeric, centromeric, and pericentromeric regions of almost all of the chromosomes, with no significative differences among cytotypes (Table 3, Figures 2 and S1-S7). Otherwise, the (CGG)10 microsatellite motif was found on chromosome pair 8 with secondary constrictions (i.e., nucleolus organizer region (NOR) carriers) in all cytotypes, and in a few additional chromosomal pairs other than in cytotype F. Additionally, (CGG)10 represents the only motif that did not accumulate in the sex chromosomes (Table 3, Figure 2). The (CA) 15 microsatellite shows hybridization signals spread over the entire length of the chromosomes, mainly in the largest ones ( Figure 2B,C). However, the signals varied according to cytotypes. Cytotypes from the north of the geographic distribution present a different distribution when compared to cytotypes from the southern distribution. This difference can be seen, for example, when the distribution of (CA) 15 in the cytotype A (50a) is compared with the distribution in the cytotype G (50b). Cytotype A visibly presents more and larger (CA) 15 blocks, in contrast to cytotype G (Table 3, Figure 2B and Figure 2C, respectively, and Figures S1-S7).
On the other hand, the microsatellites (C) 30 , (GAG) 10 , (CAG) 10 , and (CAC) 10 show a spread distribution, with a preferential accumulation in the telomeric, centromeric, and pericentromeric regions of almost all of the chromosomes, with no significative differences among cytotypes (Table 3, Figures 2 and S1-S7). Otherwise, the (CGG) 10 microsatellite motif was found on chromosome pair 8 with secondary constrictions (i.e., nucleolus organizer region (NOR) carriers) in all cytotypes, and in a few additional chromosomal pairs other than in cytotype F. Additionally, (CGG) 10 represents the only motif that did not accumulate in the sex chromosomes (Table 3, Figure 2).
The motifs (GA) 15 and (A) 30 (Figures 2A, 3 and 4, respectively) are in the telomeric, centromeric, and pericentromeric regions of most of the chromosomes, with more extensive blocks in the largest chromosome pairs of karyotypes, in all cytotypes (Table 3 and Figures S1-S7). No cytotype-specific signals appear to occur for these motifs. In summary, at least six microsatellite motifs are accumulated in near all chromosomes involved in rearrangements previously described and directly involved in the origin of the different cytotypes, as shown in    The motifs (GA)15 and (A)30 (Figures 2A, 3 and 4, respectively) are in the telomeric, centromeric, and pericentromeric regions of most of the chromosomes, with more extensive blocks in the largest chromosome pairs of karyotypes, in all cytotypes (Table 3 and Figures S1-S7). No cytotype-specific signals appear to occur for these motifs. In summary, at least six microsatellite motifs are accumulated in near all chromosomes involved in re- Concerning LINE-1-like repeats, in general, few hybridization signals were observed, with a preferential location in one of the distal chromosomal regions: in five pairs in cytotypes A, F, and G, in four pairs in cytotype B, in three pairs in cytotypes C and E, and in only two pairs in cytotype D ( Figure 5A-G).

Discussion
In mammals, chromosomal structural changes are usually related to repetitive and mobile sequences [50], especially in fragile sites [51], which consist of tandem sequences and TEs that can induce chromosomal breaks [52,53]. Indeed, in humans, primates, and mice, several EBRs have been found, evidencing that genomic reorganizations occur mainly in such regions where duplications/expansion of repetitive sequences occur [52,[54][55][56][57]. Despite the importance of repetitive sequences in chromosomal evolution, the extensive karyotype variability found in C. minutus is still poorly investigated under a molecular cytogenetic approach.
Our current data show that at least six microsatellite motifs are altered in the chromosomes of C. minutus (Figure 4), principally in centromeric and telomeric regions, whose

Discussion
In mammals, chromosomal structural changes are usually related to repetitive and mobile sequences [50], especially in fragile sites [51], which consist of tandem sequences and TEs that can induce chromosomal breaks [52,53]. Indeed, in humans, primates, and mice, several EBRs have been found, evidencing that genomic reorganizations occur mainly in such regions where duplications/expansion of repetitive sequences occur [52,[54][55][56][57]. Despite the importance of repetitive sequences in chromosomal evolution, the extensive karyotype variability found in C. minutus is still poorly investigated under a molecular cytogenetic approach.
Our current data show that at least six microsatellite motifs are altered in the chromosomes of C. minutus (Figure 4), principally in centromeric and telomeric regions, whose rearrangements were previously described [31]. The creation of loops and/or the mispairing of tandem repeats, which result in disruption-induced replication instability disorders primarily at telomeres, centromeres, and microsatellites, have been demonstrated to be associated with some neurological disorders in humans [58]. In addition, there are several examples and causes of instability of tandem repeats, owing to their intrinsic composition of sequences, secondary DNA structures, topological and physical aspects of tandem repeats, and other characteristics that can influence cellular processes and repair pathways, promoting rapid mutagenesis of the tandem repeats (see review in [59]). It has been shown that tandem repeats can mainly affect the replication mechanism during DNA synthesis, causing instability in the genome [60]. Some genomic sequences/motifs temporarily slow or stop the replication fork, increasing the likelihood of a double-strand break [61].
It is known that common fragile sites are enriched with A/T sequences and they can form secondary structures that enable chromosomal instability [52,62,63]. The A/T pairing tends to be more fragile, not only because of the number of connections, but also because of the stacking of bases [64]. Although specific repetitive sequences have not yet been seen as uniquely responsible for karyotype instability, our results showed that different microsatellite DNA motifs are found at the breakpoints in C. minutus, as observed with the monomer (A) 30, which is localized in all breakpoints of all cytotypes (Figures 2A-G, 3 and 4).
A differential distribution of the LINE-1-like element was observed for each cytotype. It is more accumulated in cytotypes A, F, and G (larger 2n) than in others ( Figure 5A-G), with cytotype D (smallest 2n) having the lowest number of signals. (Figure 5A-G). Thus, we observed that there is an association between the number of LINE-1-like signals and 2n, an association similar to the one that occurs between 2n and the geographic distribution of the species, where the extremities have higher numbers and there is a progressive decrease along the distribution and then a further progressive increase ( Figure 5). Here, we suggest that such an uneven distribution may be linked to a vertical transmission of LINE-1-like signals to all populations of C. minutus, since the presence of this retroelement precedes the divergence of marsupials and eutherians [17]. Considering that the LINE-1 element was present in a Ctenomys ancestor, it is likely that it has mutated, progressively producing fewer active copies. As a result, the occurrence of retroelements is decreasing in the populations and, probably, undergoing an extinction process in Ctenomys.
Our results also indicate that the number of LINE-1-like elements has an association with the diploid number of cytotypes ( Figure 5A-G). Our results do not suggest that L1 hybridization regions on the chromosomes are breakpoints, but rather that the lack of L1 may have provided the ideal environment for such breaks. Considering the variation in the number of signals between the different cytotypes seen in our FISH experiments ( Figure 5A-G) and that LINE-1 plays an important role in the DNA repair [66], we raised the hypothesis: considering that the cytotype A is more similar to the ancestor of C. minutus [29], the LINE-1 sequences were possibly lost or are very degenerated in the other cytotypes, thus favoring chromosomal rearrangements and the emergence of other cytotypes. Previous studies have also reported that the loss or inactivation of LINE-1 increased the chromosomal diversity, as found in the rodents of the Muroid group [70], and Oryzomys and Holochilus [49]. In addition, species with large numbers of sequenced genomes, e.g., humans from different geographic origins, showed drastic variation in the activity of some L1-like elements, contributing to human genetic variability [71]; all these observations corroborate our hypothesis.

Conclusions
This study provided evidence for a direct spatial correlation between the repetitive DNA and EBRs, highlighting their direct impact in promoting chromosomal rearrangements and the divergence of cytotypes of C. minutus. Recently, similar observations were made for songbirds of genus Senna [72]. However, although the first step towards understanding the coevolution between LINE-1 and the host genome has also been taken, it is still necessary to deepen the knowledge about the structural characteristics of LINE-1 copies, such as Southern blot, ORF isolation, and the estimation of the age of the youngest elements, to better understand this question within the Ctenomys model.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12162091/s1, Figure S1: Fluorescence in situ hybridization experiments with motif probes in cytotype A of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure S2: Fluorescence in situ hybridization experiments with motif probes in cytotype B of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure S3: Fluorescence in situ hybridization experiments with motif probes in cytotype C of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure S4: Fluorescence in situ hybridization experiments with motif probes in cytotype D of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure S5: Fluorescence in situ hybridization experiments with motif probes in cytotype E of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure S6: Fluorescence in situ hybridization experiments with motif probes in cytotype F of Ctenomys minutus. Probes used are indicated in the lower right corner of the images and the cytotype in the lower left corner of the images. Figure