E-Mail karger@karger.com Original Article Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 Chromosomal Mapping of Repetitive DNAs in Characidium (Teleostei, Characiformes): Genomic Organization and Diversification of ZW Sex Chromosomes Priscilla C. Scacchetti a Ricardo Utsunomia a José C. Pansonato-Alves a Marcelo R. Vicari b Roberto F. Artoni b Claudio Oliveira a Fausto Foresti a a Departamento de Morfologia, Universidade Estadual Paulista, Botucatu , and b Departamento de Biologia Estrutural, Molecular e Gené tica, Universidade Estadual de Ponta Grossa, Ponta Grossa , Brazil C . vidali and C . gomesi . In contrast, the number of 5S rDNA- bearing chromosomes varied. Notably, minor ribosomal clusters were identified in the W chromosome of C . vidali . Microsatellites were widely distributed across almost all chromosomes of the karyotypes, with a greater accumula- tion in the subtelomeric regions. However, clear differences in the abundance of each motif were detected in each spe- cies. In addition, the Z and W chromosomes showed the dif- ferential accumulation of distinct motifs. Our results re- vealed variability in the distribution of repetitive DNA se- quences and their possible association with sex chromosome diversification in Characidium species. © 2015 S. Karger AG, Basel Eukaryotic genomes are composed of a substantial amount of repetitive DNA sequences, such as multigene families, microsatellites (or simple sequence repeats; SSRs), and transposable elements, which play important roles in structural genomic organization [López-Flores and Garrido-Ramos, 2012]. In addition, these sequences tend to accumulate in specific chromosomes, including sex chromosomes, primarily because of their nonrecom- bining nature [Lohe et al., 1993; Kubat et al., 2008; Po- Key Words Chromosome painting · Crenuchidae · Fish cytogenetics · Karyotype diversification Abstract The speciose neotropical genus Characidium has proven to be a good model for cytogenetic exploration. Representa- tives of this genus often have a conserved diploid chromo- some number; some species exhibit a highly differentiated ZZ/ZW sex chromosome system, while others do not show any sex-related chromosome heteromorphism. In this study, chromosome painting using a W-specific probe and com- parative chromosome mapping of repetitive sequences, including ribosomal clusters and 4 microsatellite motifs – (CA) 15 , (GA) 15 , (CG) 15 , and (TTA) 10 –, were performed in 6 Characidium species, 5 of which possessed a heteromorphic ZW sex chromosome system. The W-specific probe showed hybridization signals on the W chromosome of all analyzed species, indicating homology among the W chromosomes. Remarkably, a single major rDNA-bearing chromosome pair was found in all species. The 18S rDNA localized to the sex chromosomes in C . lanei , C . timbuiense and C . pterostictum , while the major rDNA localized to one autosome pair in Accepted: June 3, 2015 by M. Schmid Published online: July 31, 2015 Priscilla C. Scacchetti Departamento de Morfologia, Universidade Estadual Paulista Distrito de Rubiã o Junior, s/n Botucatu, SP 18618-970 (Brazil) E-Mail pcardim   @   ibb.unesp.br © 2015 S. Karger AG, Basel 1424–8581/15/1462–0136$39.50/0 www.karger.com/cgr D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Repetitive DNAs in Characidium Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 137 korná et al., 2011; Kejnovský et al., 2013; Milani and Cab- ral-de-Mello, 2014; Ruiz-Ruano et al., 2015; Ziemniczak et al., 2014]. In this context, cytogenetic studies have pro- vided a better characterization of sex chromosomes by identifying distinctive degrees of heterochromatinization and differential accumulation of repetitive sequences [for a review, see Cioffi et al., 2010]. Such findings opened op- portunities for the examination of sex chromosome ho- mology and origin, investigation of sex chromosome dif- ferentiation among congeneric species, and the charac- terization of putative nascent sex chromosomes [Henning et al., 2008; Vicari et al., 2008; Cioffi and Bertollo, 2010; Schemberger et al., 2011]. Characidium comprises ∼ 70 valid species widely dis- tributed throughout neotropical rivers [Eschmeyer, 2015]. This group is an excellent model for studies of sex chromosome differentiation because some species pre- sent a highly differentiated ZZ/ZW sex system, while oth- ers do not have any heteromorphic sex chromosomes. At present, Characidium sex chromosomes are thought to have a single origin, and the proto-sex chromosome was likely NOR bearing [Vicari et al., 2008; Machado et al., 2011; Pansonato-Alves et al., 2014; Pucci et al., 2014]. Subsequently, distinct events such as an increase/de- crease in size, NOR translocation and heterochromatini- zation have shaped sex chromosome diversification in each Characidium species/population [Vicari et al., 2008; Pansonato-Alves et al., 2010, 2011a, b; Machado et al., 2011; Pucci et al., 2014]. In this study, we used a W-specific probe to test the hypothesis of a common sex chromosome origin in 6 Characidium species. In addition, we performed cytoge- netic mapping of ribosomal RNA genes and 4 distinct mi- crosatellite motifs to compare the degree of repetitive DNA accumulation on the ZW sex chromosomes with distinct degrees of heterochromatinization. The results obtained here will contribute to a better understanding of rDNA and microsatellite distribution in eukaryotic ge- nomes and will provide insight into diversification of sex chromosomes in Characidium . Materials and Methods Materials and Chromosome Banding Six allopatric Characidium species were analyzed, including C . cf. zebra , C . gomesi , C . lanei , C . timbuiense , C . vidali , and C . pterost- ictum ( table 1 ). Cell suspensions from all species were available in our laboratory, and their karyotypes were previously described [Pansonato-Alves et al., 2010, 2011a, b; Scacchetti et al., 2015]. C- banding procedures were performed as described by Sumner [1972]. Probe Preparation Oligonucleotide probes containing microsatellite sequences (CA) 15 , (GA) 15 , (CG) 15 , and (TTA) 10 were directly labeled with TAMRA during synthesis by Sigma, as described by Kubat et al. [2008]. The 5S and 18S rDNA probes were generated by PCR us- ing previously described primers [White et al., 1990; Pendás et al., 1994]. In addition, a W chromosome probe isolated from C . gomesi (CgW) was used [Pansonato-Alves et al., 2014]. The 5S rDNA was labeled with biotin-16-dUTP, while the 18S rDNA and the W chromosome probes were labeled with digoxigenin 11- dUTP. Fluorescence in situ Hybridization FISH was performed on 2 individuals of each species (1 male and 1 female). Cytogenetic mapping of ribosomal sites in C . gome- si and C . cf. zebra was described previously [Pansonato-Alves et al., 2011a, b]. The prehybridization conditions were the same for each sample as described by Pinkel et al. [1986]. Slides were incu- bated with RNAse (50 μg/ml) for 1 h at 37   °   C, and chromosomal DNA was denatured in 70% formamide/2× SSC for 5 min at 70   °   C. For each slide, 30 μl hybridization solution containing 200 ng la- beled probe, 50% formamide, 2× SSC, and 10% dextran sulfate was denatured for 10 min at 95   °   C, dropped onto the slides and hybrid- Table 1. Characidium species and populations analyzed Species Coordinates Collection sites LBP Sample size ♀ ♂ C. cf. zebra 23°30′40′′S 45°51′32′′W Paraitinga River, Salesó polis, SP 8,704 3 1 C. vidali 22°28′51.79′′S 42°23′39.06′′W Bananeiras Stream, Silva Jardim, RJ 19,039 7 2 C. gomesi 23°01′26′′S 48°49′32′′W Novo River, Avaré, SP 6,377 5 3 C. pterostictum 28°38′43.9′′S 53°33′35.7′′W Jacuí River, Cruz Alta, RS 14,672 7 8 C. timbuiense 19°58′32.56′′S 40°32′53.15′′W Valsugana Velha Stream, Santa Teresa, ES 18,475 8 7 C. lanei 25°26′29′′S 48°32′28′′W Cari River, Morretes, PR 8,700 1 2 LBP = Laboratório de Biologia e Genética de Peixes, voucher numbers; ES = Espírito Santo state; PR = Paraná state; RJ = Rio de Janeiro state; RS = Rio Grande do Sul state; SP = São Paulo state. D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Scacchetti/Utsunomia/Pansonato-Alves/ Vicari/Artoni/Oliveira/Foresti Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 138 ized overnight at 37   °   C in a 2× SSC moist chamber. Posthybridi- zation washes were performed according to the applied probe: (i) slides probed with ribosomal sites and CgW were washed in 0.2× SSC/15% formamide for 20 min at 42   °   C, followed by a second wash in 0.1× SSC for 15 min at 60   °   C, and a final wash at room temperature in 4× SSC/0.5% Tween for 10 min; probe detection was performed using avidin-FITC and anti-digoxigenin-rhoda- mine; (ii) slides probed with oligonucleotides were washed in 2× SSC for 5 min, followed by a second wash in 1× PBS for 1 min. Chromosomes were counterstained with DAPI (Vector Laborato- ries, Burlingame, Calif., USA) and analyzed using an optical pho- tomicroscope (Olympus BX61). Images were acquired using Image Pro Plus 6.0 software (Media Cybernetics, Rockville, Md., USA), and chromosomes were classified as metacentric, submeta- centric or acrocentric according to Levan et al. [1964]. Results Karyotypes The 6 species exhibited an invariable diploid chromo- some number of 2n = 50, and karyotypes were composed exclusively of bi-armed chromosomes, except C . tim- buiense and C . pterostictum , where the karyotypes con- tained one acrocentric pair ( fig. 1 ). Microsatellite Distribution and C-Banding In general, FISH experiments using the probes (CA) 15 , (GA) 15 , (CG) 15 , and (TTA) 10 showed the same distribu- tion pattern in autosomes with a preferential accumula- tion in subtelomeric regions, except for the motif (CG) 15 in C . timbuiense and C . cf. zebra , which apparently was restricted to a single chromosome pair ( fig. 1 ). C-banding identified the ZW sex chromosomes in all species, except C . cf. zebra ( fig. 2 ). Notably, the sex chro- mosomes displayed different degrees of heterochromati- nization. For example, the C . gomesi W chromosome was almost fully heterochromatic, while the W chromosome of the remaining species was partially heterochromatic ( fig. 2 ). We observed distinct patterns of SSR distribution on the Z and W chromosome of each species. These se- quences localized to both euchromatic and heterochro- matic areas of the sex chromosomes, including subtelo- meric and interstitial regions. We also observed that the C . gomesi W chromosome showed intense accumulation of (TTA) 10 along its entire length ( fig. 2 ). Ribosomal Probes FISH analyses with an 18S rDNA probe revealed sig- nals in the subtelomeric regions of the Z and W chromo- somes of C . timbuiense , C . lanei and C . pterostictum, while the major ribosomal sites were located to one autosomal pair in C . vidali and C . gomesi and in chromosome pair 23 in C . cf. zebra ( figs. 2 , 3 ). We observed 5S rDNA probe hybridization signals in 1 autosomal pair of C . lanei , and 3 autosomal pairs in C . pterostictum and C . timbuiense . In C . vidali , we detected signals in the W chromosome and in 1 autosomal pair ( figs. 2 , 3 ). Whole Chromosome Painting The CgW probe painted the entire C . gomesi W chro- mosome and the pericentromeric region of the Z chro- mosome. In C . lanei and C . pterostictum, the CgW probe hybridized to the long arm of the W chromosome and in the pericentromeric region of the Z chromosome. Finally, the probe painted the long arm of the W chromosome and the pericentromeric and peritelomeric region of the Z chromosome of C . vidali ( figs. 2 , 4 ). Discussion In this study, we characterized the chromosomal dis- tribution of microsatellite motifs in Characidium . In gen- eral, both di- and trinucleotides were widely and simi- larly distributed in the genomes of the analyzed species, with a preferential accumulation in subtelomeric areas. In fact, it has been postulated that microsatellite accumula- tion in eukaryotic genomes is nonrandom and that each group of organisms/species show preferential accumula- tion of specific SSR motifs with a particular chromosom- al distribution [Tóth et al., 2000; Ruiz-Ruano et al., 2015]. The (CA) 15 , (GA) 15 and (CG) 15 SSRs were previously mapped in different characiform fishes and had similar subtelomeric distributions in Hoplias , Leporinus and Tri- portheus species, while 2 Semaprochilodus species showed few centromeric/subtelomeric signals [Cioffi et al., 2011, 2012; Poltronieri et al., 2013; Terencio et al., 2013; Yano et al., 2014]. Although there is a clear tendency of SSRs to accumu- late in subtelomeric regions of Characidium chromo- somes, it must be noted that microsatellite distribution and accumulation in autosomes is susceptible to changes at the interspecies level and at the intragenome level. For example, the (CG) 15 motif clustered to the subtelomeric Fig. 1. FISH mapping of microsatellite motifs in Characidium spe- cies. Chromosomes were counterstained with DAPI (blue), and microsatellite probes were directly labeled with TAMRA (red). Arrows indicate acrocentric chromosomes. Z, W = Sex chromo- somes; B = B chromosomes. Scale bar = 10 μm. (For figure see next page.) D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Repetitive DNAs in Characidium Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 139 1 D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Scacchetti/Utsunomia/Pansonato-Alves/ Vicari/Artoni/Oliveira/Foresti Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 140 Fig. 2. Z and W chromosomes of Chara- cidium species after Giemsa, C-banding and FISH with the CgW probe; 18S (red) and 5S rDNA (green) probes and microsat- ellite probes (CA) 15 , (CG) 15 , (GA) 15 , and (TTA) 10 directly labeled with TAMRA (red). Fig. 3. Metaphase chromosome plates after FISH with 5S (green) and 18S rDNA (red) probes. Z, W = Sex chromosomes. Scale bar = 10 μm. D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Repetitive DNAs in Characidium Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 141 regions of almost all chromosomes in C . gomesi , C . lanei , C . pterostictum , and C . vidali , while the signal was re- stricted to 1 or 2 chromosomes in C . timbuiense and C . cf. zebra ( fig. 1 ). Because previous studies have shown that C .cf. zebra and C . timbuiense are not closely related spe- cies [Buckup, 1993; Vicari et al., 2008; Pansonato-Alves et al., 2014, Scacchetti et al., 2015], we suggest that this specific SSR was partially and independently eliminated from the genomes of both species, suggesting that chro- mosomal distribution of microsatellite motifs in Chara- cidium does not reflect the evolutionary history of associ- ated taxa. In addition, we observed remarkable interchro- mosomal differences in SSR abundance. These results are consistent with the idea that SSR genomic organization is governed by distinct molecular mechanisms, including ectopic recombination, unequal crossing-over, slippage replication and association with transposable elements. Together, these mechanisms control the expansion, ac- cumulation and elimination of these sequences at differ- ent levels of resolution (e.g. interspecific, interpopula- tional and/or intragenome) [Dover, 1993; McMurray, 1995; Hancock, 1996, Milani and Cabral-de-Mello, 2014; Ruiz-Ruano et al., 2015]. We also investigated the genomic location of minor and major ribosomal DNAs in the C . vidali , C . timbuiense and C . lanei species. Our results are consistent with the general patterns of chromosomal distribution of ribo- somal genes in genomes of representatives of this genus, with two 18S rDNA clusters per genome and variable 5S rDNA clusters per genome. Such patterns have been de- scribed for several Characidium species, and their dy- namics have been attributed to the association with trans- posable elements [Pucci et al., 2014], as described for many other fish species [da Silva et al., 2011; Silva et al., 2013]. The CgW probe hybridized to the sex chromosomes of the other analyzed species, consistent with the hypothesis that the Characidium ZZ/ZW sex chromosomes are ho- mologous among different species [Machado et al., 2011, Pazian et al., 2013; Pansonato-Alves et al., 2014; Pucci et Fig. 4. Metaphase chromosome plates after FISH with CgW probe. Z, W = Sex chro- mosomes. Scale bar = 10 μm. D ow nl oa de d by : U N E S P U ni ve rs id ad e E st du al P au lis ta 18 6. 21 7. 23 6. 55 - 6 /1 8/ 20 19 6 :3 7: 10 P M http://dx.doi.org/10.1159%2F000437165 Scacchetti/Utsunomia/Pansonato-Alves/ Vicari/Artoni/Oliveira/Foresti Cytogenet Genome Res 2015;146:136–143 DOI: 10.1159/000437165 142 al., 2014]. Although they have been suggested to have a common origin, ZW chromosome diversification in dif- ferent species is remarkable. Previous studies have suggested that the presence of 18S rDNA sites in Characidium sex chromosomes imply an intermediate degree of heterochromatinization of the W chromosome. However, if the NOR was translocated to an autosome, the W chromosome would show a high degree of heterochromatinization [Vicari et al., 2008; Pansonato-Alves et al., 2010; Machado et al., 2011; Pucci et al., 2014]. Such a feature is based on the premise that the NOR-bearing sex chromosomes could still exchange chromosomal segments, thus reducing the rates of evolu- tionary differentiation. Accordingly, Z and W chromo- somes without NOR sites would result in decreased re- combination frequency and, consequently, favor their differentiation (e.g. the heterochromatinization process) [Pansonato-Alves et al., 2011a, b]. Our results corrobo- rate this hypothesis, with the exception of C . vidali , which exhibited Z and W chromosomes in intermediate stages of heterochromatinization, although they do not display NOR sites. Interestingly, the C . vidali W chromosome also shows an exclusive 5S rDNA cluster, which could even further decrease the recombination frequency be- tween heteromorphic chromosomes. Therefore, we hy- pothesize that NOR translocation occurred more recent- ly in C . vidali than in C . gomesi, and its sex chromosomes are still in the intermediate developmental stages of dif- ferentiation. Remarkably, minor ribosomal sequences in the C . vidali W chromosome are highly derived charac- teristics of this genus, unlike the 18S rDNA. SSR chromosomal mapping revealed well-differentiat- ed ZZ/ZW sex chromosomes in all species with different repetitive DNA class accumulation in distinct regions. For example, some motifs may be more abundant in the Z chromosome, while others may be exclusively localized to the W chromosome, such as (CG) 15 in C . timbuiense . Therefore, we suggest that at a specific period of time, a particular microsatellite might expand and become fixed in the sex chromosomes due to their nonrecombining na- ture. The lack of consistency for SSR accumulated in the sex chromosomes of different species likely reflects his- torical contingency as proposed by Pokorná et al. [2011] and should occur stochastically. In all these cases, it is clear that the differential SSR distribution may further decrease the recombination frequency between the Z and W chromosomes and, consequently, play a role in their differentiation process. In general, we did not observe preferential SSR accu- mulation in W chromosomes. An exception is C . gomesi , which showed conspicuous (TTA) 10 motif accumulation along the entire W chromosome length. Coincidently, it is also the only species in our study that exhibited a fully heterochromatic W chromosome, suggesting an associa- tion between heterochromatinization and (TTA) 10 accu- mulation. Consistent with this result, previous studies have shown greater SSR accumulation in the heterochro- matic areas of the sex-specific chromosome [Pokorná et al., 2011; Cioffi et al., 2012; Poltronieri et al., 2013; Teren- cio et al., 2013; Nanda et al., 2014; Yano et al., 2014; Ziem- niczak et al., 2014]. However, we found that all other SSRs clustered to either euchromatic or heterochromatic re- gions of the sex chromosomes, indicating that they are not necessarily associated with heterochromatin accu- mulation. In the present study, the large diversity of SSR chro- mosomal distribution patterns in Characidium species provides new evidence for the intense and continuous ge- nomic changes in closely related species of the genus. 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