Chromosoma https://doi.org/10.1007/s00412-018-0666-9 ORIGINAL ARTICLE High-throughput analysis of satellite DNA in the grasshopper Pyrgomorpha conica reveals abundance of homologous and heterologous higher-order repeats Francisco J. Ruiz-Ruano1 & Jesús Castillo-Martínez1,3 & Josefa Cabrero1 & Ricardo Gómez2 & Juan Pedro M. Camacho1 & María Dolores López-León1 Received: 7 September 2017 /Revised: 13 February 2018 /Accepted: 6 March 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Satellite DNA (satDNA) constitutes an important fraction of repetitive DNA in eukaryotic genomes, but it is barely known in most species. The high-throughput analysis of satDNA in the grasshopper Pyrgomorpha conica revealed 87 satDNA variants grouped into 76 different families, representing 9.4% of the genome. Fluorescent in situ hybridization (FISH) analysis of the 38 most abundant satDNA families revealed four different patterns of chromosome distribution. Homology search between the 76 satDNA families showed the existence of 15 superfamilies, each including two or more families, with the most abundant superfamily representing more than 80% of all satDNA found in this species. This also revealed the presence of two types of higher-order repeats (HORs), one showing internal homologous subrepeats, as conventional HORs, and an additional type showing non-homologous internal subrepeats, the latter arising by the combination of a given satDNA family with a non- annotated sequence, or with telomeric DNA. Interestingly, the heterologous subrepeats included in these HORs showed higher divergence within the HOR than outside it, suggesting that heterologous HORs show poor homogenization, in high contrast with conventional (homologous) HORs. Finally, heterologous HORs can show high differences in divergence between their constit- uent subrepeats, suggesting the possibility of regional homogenization. Keywords FISH . Higher-order repeat (HOR) . High-throughput sequencing . Pyrgomorpha conica . Satellitome Introduction the most abundant repeated sequences and constitutes a major component of heterochromatin in numerous species of plant A substantial part of eukaryotic genomes is composed of dif- and animals (Charlesworth et al. 1994). In eukaryotes, these ferent repeated sequences (Britten and Kohne 1968;López- sequences can represent up to half of its genome content Flores and Garrido-Ramos 2012; Garrido-Ramos 2017). (Plohl et al. 2012). Among them, satellite DNA (satDNA) is considered one of SatDNA consists in a non-genic repeat unit (RU) of a given length which appears tandemly repeated and organized in ar- rays of variable length and complexity. They can be classified Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00412-018-0666-9) contains supplementary into microsatellites, minisatellites and satellites according to material, which is available to authorized users. RU length (RUL), although there is no agreement on the length thresholds to delimit these three subclasses. A frequent * María Dolores López-León convention is 1–6, 7–100 and > 100 bp for micro-, mini- and [email protected] satellites, respectively. In addition, micro- and minisatellites are frequently considered to be rarely locally amplified thus 1 Departamento de Genética. Facultad de Ciencias, Universidad de failing to show bands visible by FISH on chromosomes, thus Granada, 18071 Granada, Spain being considered as genomic elements being scattered across 2 Departamento de Ciencia y Tecnología Agroforestal, E.T.S. de the genome. However, satellites (sometimes called Ingenieros Agrónomos, Universidad de Castilla La Mancha, 02071 Albacete, Spain macrosatellites, see Kass and Batzer 2001) are usually consid- ered to be locally amplified thus showing visible bands on 3 Present address: Facultad de Medicina, Universidad Católica de Valencia, C/Quevedo 2, 46001 Valencia, Spain chromosomes. These conventions have recently been Chromosoma overturned after the high-throughput analysis of satDNA cat- data in species such as the grasshoppers Schistocerca gregaria alog (i.e., the satellitome) in the grasshoppers Locusta (Camacho et al. 2015a), L. migratoria (Ruiz-Ruano et al. migratoria (Ruiz-Ruano et al. 2016)andEumigus monticola 2016)and E. monticola (Ruiz-Ruano et al. 2017) has enabled (Ruiz-Ruano et al. 2017), since satDNAs showing both long the detection of satDNAs that remained elusive by traditional or short RUs can be visible or not by FISH. In fact, the exis- methods. This has facilitated the global analysis of the whole tence of satDNAs showing short RUs, fitting the conventional satDNA catalog, i.e., the satellitome, which, in the case of definition of micro- and minisatellites, but displaying conspic- L. migratoria, unveiled 62 satDNA families and provided uous bands after in situ hybridization, had previously been new insights about the origin and evolution of these intriguing reported in other organisms, such as Musca domestica sequences. (Blanchetot 1991), Drosophila melanogaster (Bonaccorsi Chromosome location of satDNA is prevalent on centro- and Lohe 1991) or grasshoppers (Ruiz-Ruano et al. 2015). meric and telomeric regions (Charlesworth et al. 1994)in Recent findings have also shown that satDNA is not restricted coincidence with heterochromatin location, although it can to heterochromatin (Ruiz-Ruano et al. 2016, 2017). Therefore, also be found on interstitial euchromatic regions (Ruiz- the most inclusive definition of satDNA would be one com- Ruano et al. 2016). Only a few satDNAs are located on all prising only universal properties of satDNA, such as its tan- chromosomes within a genome, whereas other are dem repeat structure and its non-genic character (to differen- chromosome-specific (Plohl et al. 1998; Ruiz-Ruano et al. tiate it from tandem repeat gene families). 2016, 2017). In several insect species, satDNA being specific Traditionally, satDNA has been identified and isolated of X or B chromosomes have also been found (for review, see through restriction endonuclease digestion of genomic DNA, Palomeque and Lorite 2008; López-Flores and Garrido- monomer cloning and Sanger sequencing. However, this ap- Ramos 2012; Garrido-Ramos 2017; Ruiz-Ruano et al. 2017). proach limits the number of satDNA families that can be char- The grasshopper Pyrgomorpha conica is a non-model spe- acterized to those most represented in the genome, whereas cies with very limited molecular infomation on its chromo- many low-abundance satDNAs go unnoticed. Nowadays, the somes. Classical cytogenetic studies have described its karyo- arrival and improvement of next generation sequencing type as composed of nine pairs of autosomes that can be clas- (NGS) technologies (van Dijk et al. 2014) has provided an sified into large (L1-L3), medium (M4-M8) and small (S9) opportunity for cloning-free massive sequencing of genomes sized chromosomes, plus one or two X chromosomes in males which has yielded huge volumes of genomic data on different or females respectively. All chromosomes are apparently telo- species, including the repeat fraction of genomes that previ- centric, and the S9 autosome behaves as the megameric biva- ously appeared underrepresented in the genome sequencing lent during meiosis, as it is differentially condensed through- projects. In fact, satDNA is scarcely mentioned in works de- out first profase (Antonio et al. 1993). The scarce knowledge scribing whole genome sequences. In human, for instance, the concerning the molecular nature of chromosomes of this spe- longest assembly gaps correspond to satDNA-rich centromere cies is restricted to the presence of large heterochromatin regions and the short arm of acrocentric chromosomes (for blocks on pericentromeric regions of all chromosomes review, see Miga 2015). However, studies on repetitive (Santos et al. 1983), the presence of sites sensitive to restric- DNA, and particularly on satDNA, are of high interest to tion endonucleases on them (López-Fernández et al. 1988), completely understand genome structure and dynamics. the physical mapping of ribosomal DNA (rDNA) on the M5 Additionally, during the last three decades, the number of and M6 chromosomes (Suja et al. 1993) and the identification evidences pointing to a functional role of these sequences of interstitial telomeric DNA regions (ITRs) on many chro- has steadily increased, and satDNA has been found to be mosomes (López-Fernández et al. 2004, 2006). involved in the establishment of heterochromatic domains SatDNA units are sometimes simple, i.e., composed of a (Henikoff 1998; Hsieh and Fire 2000), centromere function single sequence with no internal substructuring. However, (Plohl et al. 2014), chromosome paring and segregation (Lica there are also complex satDNAs with units showing internal et al. 1986;John1988), nuclear architecture (Hemleben et al. duplication of a subunit, which may be inverted or not, and the 2000) or gene expression (Feliciello et al. 2015). As a conse- eventual insertion of foreign DNA (Charlesworth et al. 1994; quence, NGS and bioinformatic graph-based methods (Novák Meštrović et al. 2015). SatDNA repeats sometimes combine et al. 2013) are now preferentially used to identify and char- into higher-order repeats (HORs) composed of different vari- acterize the repetitive DNA fraction of genomes in a variety of ants of a same satDNA family, which are better homogenized animal and plant species (Macas et al. 2007, 2011; Novák than the constituent subrepeats (Willard and Waye 1987; et al. 2014;Garcíaetal.2015).