Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 111-119, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1296 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Simona Ceraulo, Vanessa Milioto, Francesca Dumas (2021) Cen- tromeric enrichment of LINE-1 retro- transposon in two species of South American monkeys Alouatta belzebul and Ateles nancymaae (Platyrrhini, Pri- mates). Caryologia 74(4): 111-119. doi: 10.36253/caryologia-1296 Received: April 22, 2021 Accepted: November 27, 2021 Published: March 08, 2022 Copyright: © 2021 Simona Ceraulo, Vanessa Milioto, Francesca Dumas. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Centromeric enrichment of LINE-1 retrotransposon in two species of South American monkeys Alouatta belzebul and Ateles nancymaae (Platyrrhini, Primates) Simona Ceraulo, Vanessa Milioto, Francesca Dumas* Department of Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, Palermo, Italy *Corresponding author. E-mail: francescadumas@unipa.it Abstract. LINE-1 sequences have been linked to genome evolution, plasticity and speciation; however, despite their importance, their chromosomal distribution is poorly known in primates. In this perspective, we used fluorescence in situ hybridiza- tion (FISH) to map LINE-1 probes onto two representative platyrrhine species, Aotus nancymaae (Cebidae) and Alouatta belzebul (Atelidae), both characterized with high- ly rearranged karyotypes, in order to investigate their chromosomal distribution and role and to better characterize the two genomes. We found centromeric enrichment of LINE-1 sequences on all biarmed and acrocentric chromosomes co-localized with heterochromatin C-positive bands. This distribution led us to hypothesize that LINE 1 sequences may have a role in the centromere architecture and karyotype organization of platyrrhine genomes. Keyword: transposable elements, C-banding, molecular cytogenetics probes, genome evolution. INTRODUCTION Through classic and molecular cytogenetics, many primates have been shown to have variable karyotypes; many kinds of probes have been mapped, including single locus probes (Dumas and Sineo 2010, 2012), and Bacterial Artificial Chromosomes (BAC) (Dumas and Sineo 2014; Dumas et al. 2015) and whole chromosome paints have been used (Dumas et al. 2007; Dumas et al. 2012), showing a high rate of intrachromosomal and intrachromosomal rearrangements. In particular, among Platyrrhini (New World primates) liv- ing in tropical and neotropical regions, the genera Alouatta (howler mon- keys) (Cebidae) and Aotus (owl monkey) (Cebidae) have very derived kary- otypes. Originally, one or only a few species were recognized in these two genera: in Aotus there was just one, while later up to eleven species were described, with many of them showing different karyomorphs and having diploid numbers ranging between 2n=46 to 56; Alouatta went from five rec- 112 Simona Ceraulo, Vanessa Milioto, Francesca Dumas ognized species up to 15, with diploid numbers ranging between 2n=43 to 58. Furthermore, both species show an extra sex chromosome system due to a translocation between an autosome and the Y chromosome. Among the two genera, chromosome painting has been applied to two Aotus species, Aotus nancymaae, and Aotus lemurinus greisemebra (Stanyon et al. 2004; Stanyon et al. 2011) and six Alouatta species, including Alouatta belzebul (Consigliere et al. 1996, 1997; de Oliveria et al. 2002), showing high genome variability. BAC-FISH has also been performed on Aotus and Alouatta showing intrachromosomal rearrangements (Dumas et al. 2015; Scardino et al. 2020a). Although, these species have been studied through molecular cytogenetics with different kinds of probes, repetitive sequences have been poorly studied and, among them, only rDNA and Telomeric probes have been mapped often (Mazzoleni et al. 2017; 2018, Ceraulo et al. 2021a). The study of these sequence probes’ distribution can help locate useful cytogenetic markers for evolutionary and phylogenetic studies. Repetitive elements have been extensively investi- gated in order to clarify their possible role in genome evolution and organization (Ahmed and Liang 2012; Biscotti et al. 2015; Dumas et al. 2016; Mazzoleni et al. 2017, 2018; Milioto et al. 2019; Paço, et al. 2019; Scar- dino et al. 2020b). In primates, repetitive sequences constitute about 50% of their genome and are linked to chromosome evolution (Mathews et al. 2003; Jurka 2007; Xing et al. 2007; Kvikstad and Makova 2010). A class of repetitive sequences called Long Interspersed Elements of the family 1 (LINE-1) are retrotranspos- able; the biological roles of this repetitive DNA frac- tion have been linked to many mechanisms implicated in the genome structure, evolution and disease (Zhu et al. 2011; Paço et al. 2019). In addition, their involve- ment in genome architecture such as in DNA packag- ing, centromere stability and plasticity, gene expression, and epigenetic mechanisms has been shown (Kim and Han 2015; Klein and O’Neill 2018; Ahmed et al. 2020). These sequences have also been supposed to be promot- ers of genomic evolutionary changes and of biological diversity among vertebrates, with an important role in speciation (Böhne et al. 2008; Belyayev 2014, Klein and O’Neill 2018). With advances in DNA technologies, the approaches useful for identifying them have changed, with the main approaches being the use of restriction enzyme digestion of DNA, in situ hybridization and bio- informatic analysis of DNA sequencing data. In mam- mals and primates, LINE-1 were studied through differ- ent approaches including the use of restriction enzymes (Seuanez et al., 1989) or whole genome screening in sim- ians (Ohshima et al. 2003). In humans and anthropoids, LINE-1 sequence comparisons have been made (Ovchin- nikov et al. 2001, 2002, Mathews et al. 2003) showing that LINE-1 amplification may change rapidly during primate evolution giving different families with variable forms Among New World monkeys, high LINE activity has also been shown in the Saimiri and Saguinus line- ages (Callitrichini), and reduced activity has been found in the Ateles lineage (Boissinot et al. 2004, Sookdeo et al. 2018). So far, however, the distribution of these repetitive sequences through FISH with LINE-1 probes has been studied in few platyrrhine species, belonging to the Cal- lithricini subfamily of the Cebidae family (Serfaty et al. 2017, Ceraulo et al. 2021b). The objective of this study was to overcome this lack and analyze the distribution of these LINE-1 sequenc- es by FISH onto two more representative platyrrhine genomes, Aotus nancymaae (Atelidae) and Alouatta belzebul (Cebidae), with the aim of contributing to the understanding of their role and dynamics as well as the evolution of these highly derived groups of species. MATERIALS AND METHODS Following the standard protocol (Scardino et al. 2020b), metaphases were obtained from primary fibro- blast cell line cultures for Aotus nancymaae and Alouat- ta belzebul. L1 Probe preparation DNA extraction from the cell culture pellet derived from the fibroblast cell line was done according to the basic DNA extraction protocol from Invitrogen. LINE- 1 retrotransposon was amplified through polymerase chain reaction (PCR) using the following primers: L1R, 5’-ATTCTRTTC CAT TGG TCT A-3’ and L1F 5’-CCA TGC TCATSGAT TGG -3’ (Waters et al. 2004). 200 ng of genomic DNA was amplified in 50 μl reac- tions in an Applied Biosystems PCR SimpliAmp thermal cycler (Thermo Fisher Scientific): five units of Taq DNA Polymerase were incubated together with the template DNA, 500 nM of each primer, 200 μM each of dATP, dCTP, dTTP and dGTP in 10 mM TRIS-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl. Cycling parameters were 30 cycles of 94°C, 30 s; 52.5°C, 30 s; 72°C, 30 s, following a 2 min denaturation at 94°C. Products were visualized on 1% agarose gel. The PCR amplification products were labelled through nick translation using 11-dUTP-Fluorescein. 113Centromeric enrichment of LINE-1 retrotransposon in Alouatta belzebul and Ateles nancymaae FISH, karyotyping and chromosome staining Fluorescence in situ hybridization (FISH) was per- formed following previously described protocols (Scardi- no et al. 2020a, b; Milioto et al. 2019; Vizzini et al. 2021); C-banding was done sequentially, post-FISH, accord- ing to a protocol which includes denaturation with for- mamide (Fernandez et al. 2002). The karyotype was reconstructed using both G-banding and inverted DAPI banding, in agreement with previous published karyo- types: Aotus nancymaae (Stanyon et al. 2004, 2011; Ruiz- Herrera et al. 2005; Dumas et al. 2016) and Alouatta belzebul (Consigliere et al. 1998; de Oliveira et al. 2002; Stanyon et al. 2011). DAPI images were inverted with a photo editing program (Adobe Photoshop); inverted gray bands correspond to dark G bands. The chromosomes with the LINE-1 probe signals were identified using inverted DAPI. After FISH, the metaphases were analyzed under a Zeiss Axio2 epifluo- rescence microscope. Images were captured using a cou- pled Zeiss digital camera, and the chromosomes were classified according to the nomenclature proposed by Levan et al. (1964). RESULTS Metaphases of the two analyzed species, obtained through cell culture and chromosome harvesting, were stained post-FISH using DAPI. The species studied here have the diploid number of 2n=50 and 2n=54, respectively, in Alouatta belzebul and Aotus nancymaae (Fig 1, 2). The former has 11 pairs of metacentric and submetacentric chromosomes (1-11) and 13 acrocentric chromosomes pairs (12-24) plus XY; the second species has 18 pairs of metacentric and submeta- centric chromosomes (1-18) plus the XX, and 7 acrocen- tric/subacrocentric chromosome pairs (19-26); C-band- ing showed signals at the centromeric position of both biarmed and acrocentric chromosomes pairs (Fig 1, 2) with peculiar amplified C bands on the bigger subtelo- centric chromosomes, in agreement with previous analy- sis (Torres et al. 1998). In all the analyzed species, the LINE-1 probe map- ping revealed bright signals at the centromeric position of almost all chromosomes, with a variable signal ampli- fication. On subtelocentric chromosomes, signals were very bright on the p arms (Fig. 1, 2). Chromosome X was rich in LINE-1 at the centromere. We did not find L1 signals in areas away from centromeres. We specu- lated that L1 elements should be in a lower copy number in chromosome regions far from centromeres, below the detection efficiency of FISH, or our probe was not able to hybridize the variable or degraded L1 elements. DISCUSSION In general, many mammalian species have deposi- tion of this LINE-1 element in euchromatic regions in G-positive bands (Parish et al. 2002; Waters et al. 2004), while in other few species it occurs in heterochromatic regions, especially in the centromeric region (Waters et al. 2004). However, the pattern of LINE-1 distribution at the centromere is not a common phenomenon among the mammalian genome (Waters et al. 2004; Dobigny et al. 2004, 2006; Acosta et al. 2008; Vieira-da- Silva et al. 2016; de Sotero-Caio et al. 2017); indeed, these elements are not often incorporated at major core centromeres, with the exception of the X chromosome euchromatic regions where they are usually abundant along the chro- mosomal length (Waters et al. 2004; Acosta et al. 2008). On the other hand, massive accumulations of repetitive elements at the centromeres was previously shown in many other mammals, such as bats and rodents (Sote- ro-Caio et al. 2017; Paco et al. 2015; Paço et al. 2019), and in some primates (Carbone et al. 2012; Serfaty et al. 2017, Ceraulo et al., 2021b). In particular, among primates species, LINE-1 have been previously identi- fied through FISH into platyrrhine genomes, in Sagui- nus midas and Saguinus bicolor (Serfaty et al. 2017), in Saguinus mystax, Leontocebus fuscicollis, Leontopithecus rosalia (Ceraulo et al., 2021b) (tamarins of the Cebidae family). This result is in agreement with sequence data anal- ysis that showed active LINE on Cebidae species (Boissi- not et al. 2004) and, more recently, also in Atelidae (Sookdeo et al. 2018). At the beginning, from an analysis of just Ateles paniscus (Boissinot et al. 2004), the extinc- tion of LINE 1 in Atelidae was proposed, but a larger phylogenetic sampling permitted researchers to show their presence (Sookdeo et al. 2018). In our work, we found LINE-1 elements by FISH in the two species analyzed of both the Cebidae and Atelidae families, at centromeric position in agreement with previous cytogenetic molecular data (Serfaty et al. 2017, Ceraulo et al., 2021b) and supporting also previ- ous molecular data (Sookdeo et al. 2018). LINE-1 probes displayed a non-random distribution by accumulating primarily in CMA3 positive bands at centromeres or pericentromeric regions, co-localizing with C-positive heterochromatin bands (Fig 1, 2); the co-localization of LINE-1 with C-positive bands was previously identified 114 Simona Ceraulo, Vanessa Milioto, Francesca Dumas Figure 1. Examples of Alouatta belzebul metaphases in DAPI blue (a), FISH with LINE-1 probe in green (b), sequential C-inverted banding (c), DAPI and LINE-1 overlap (d); the reconstructed karyotype from another metaphase of the species after sequential staining and probe mapping (e). 115Centromeric enrichment of LINE-1 retrotransposon in Alouatta belzebul and Ateles nancymaae Figure 2. Examples of Aotus nancymaae metaphases in DAPI blue (a), FISH with LINE-1 probe in green (b), and G-inverted banding (c), DAPI and LINE-1 in overlap (d); the reconstructed karyotype from the same metaphase after sequential staining and probe mapping (e). 116 Simona Ceraulo, Vanessa Milioto, Francesca Dumas not only in primates but also in other taxa (Kapitonov et al. 1998; Serfaty et al. 2017). The finding of the centro- meric enrichment of LINEs in all analyzed platyrrhine species permitted us to hypothesize that this accumula- tion might have occurred in the common ancestor of all Platyrrhini, contributing to their current karyotype fea- tures. These very intense, amplified and bright signals at centromeres possibly indicate that LINE-1, together with the alpha satellite DNA, are presumably responsible for the architecture of almost all biarmed and acrocentric chromosomes in Platyrrhini. Traditionally, alpha-satel- lite DNA has been identified as the main DNA compo- nent of primate centromeres; it consists of multiple repeat units forming a larger repeat unit, and the larger units are repeated tandemly (Koga et al. 2014), with exception of marmosets where the larger repeat unit is not pre- sent (Cellamare et al. 2009). The presence of LINE-1 at the centromere position is not a surprise; indeed, in the pericentromeric region of the human genome, for exam- ple, in addition to satellite DNA, additional elements, mainly retrotransposon elements, have also been shown (Ahmed et al. 2020). Moreover, the presence of differ- ent and diverse sequences at the centromere have also been shown in the platyrrhine genome, indicating that this region has very variable components in New World monkeys (Valeri et al. 2021). Furthermore, although transposable elements and satellite DNA present differ- ences in their structure, genomic organization, spreading mechanisms and evolutionary dynamics, several studies have highlighting their relatedness, with transitions from transposable elements to alpha-satellite DNA, and vice versa, through a process known as the DNA remodeling mechanism (Mestrovich et al. 2015; Paco et al. 2019). The possible link between transposable elements and alpha- satellite DNA, could be show in the LINE signal ampli- fication at the centromere position of subtelocentric/ acrocentric chromosome p arms, localizing with het- erochromatin C-positive bands in Ateles chromosomes; these regions are the same where often it is found telo- meric signal probes and rDNA probes amplification in many primates, as previous works have demonstrated (Mazzoleni et al. 2017, 2018, Ceraulo et al., 2021a); thus, this observation presumably indicates that these LINE- 1, alpha-satellite and other repetitive sequences could be involved to this DNA remodeling mechanism. However, considering that LINE have also been linked with chro- mosomal rearrangements (Böhne et al. 2008; de Sotero- Caio et al. 2017, Klein and O’Neill 2018), we should take into consideration that these sequences could have had a role in the process leading to the increased rates of chro- mosomal evolution responsible for the highly rearranged karyotypes of many taxa; for example, the link between evolutionary reshufflings and LI accumulation have been hypothesized in other platyhirrine species Saguinus mystax, Leontocebus fuscicollis, Leontopithecus rosalia (Ceraulo et al., 2021b) and from data obtained in rodent genera, where the most derived species display a higher level of LINE-1 accumulation on both autosomal and sex chromosomes then the more conserved ones (with accu- mulation usually only in the sex chromosomes) (Dobigny et al. 2004; Rebuzzini et al. 2009; Vieira- Dasilva et al. 2016). In order to clarify this hypothesis regarding the link between evolutionary reshufflings and LI accumulation, more samples should be analyzed in the future in a com- parative perspective considering derived and conserved primate taxa. CONCLUSION Chromosomal studies through FISH mapping onto the analyzed species’ genomes permitted the localiza- tion of the LINE sequences at the centromere position of biarmed and acrocentric chromosomes, co-localizing with C-positive bands. In a comparative perspective, the presence of these sequences at centromeres in many platyrrhine species led us to propose that LINE-1 could have had a role in the architecture and organization of the present features of platyrrhine karyotypes. In the future, studies on more samples at the species and pop- ulation level could help in understanding their origin, evolutionary dynamics and function in the karyotypes of primates. FUNDING The study was supported by an FFR 2020 grant from the University of Palermo to FD. 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BRCA1 tumour suppression occurs via heterochro- matin-mediated silencing. Nature, 477, 179–184. DOI: 10.1038/nature10371 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 4 - 2021 Firenze University Press Cytogenetic analyses in three species of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina) Kevin I. Sánchez1,*, Fabio H. Takagui2, Alberto S. Fenocchio3 Genetic variations and interspesific relationships in Lonicera L. (Caprifoliaceae), using SCoT molecular markers Fengzhen Chen1, Dongmei Li2,* , Mohsen Farshadfar3 The new chromosomal data and karyotypic variations in genus Salvia L. 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