Satellitome Analysis in the Ladybird Beetle Hippodamia variegata (Coleoptera, Coccinellidae)

Hippodamia variegata is one of the most commercialized ladybirds used for the biological control of aphid pest species in many economically important crops. This species is the first Coccinellidae whose satellitome has been studied by applying new sequencing technologies and bioinformatics tools. We found that 47% of the H. variegata genome is composed of repeated sequences. We identified 30 satellite DNA (satDNA) families with a median intragenomic divergence of 5.75% and A+T content between 45.6% and 74.7%. This species shows satDNA families with highly variable sizes although the most common size is 100–200 bp. However, we highlight the existence of a satDNA family with a repeat unit of 2 kb, the largest repeat unit described in Coleoptera. PCR amplifications for fluorescence in situ hybridization (FISH) probe generation were performed for the four most abundant satDNA families. FISH with the most abundant satDNA family as a probe shows its pericentromeric location on all chromosomes. This location is coincident with the heterochromatin revealed by C-banding and DAPI staining, also analyzed in this work. Hybridization signals for other satDNA families were located only on certain bivalents and the X chromosome. These satDNAs could be very useful as chromosomal markers due to their reduced location.


Introduction
Eukaryotic genomes are composed of single copy sequences and repetitive sequences that are mainly responsible for the structure, size and genome diversity [1]. The repeated DNA is composed of transposable elements and tandem repetitions [2]. The most significant portion in a eukaryotic genome is composed of repetitive DNA sequences tandemly repeated among the genome, known as satellite DNA (satDNA), and discovered in cesium-chloride density gradients [3]. They are constituted by highly repeated sequences with a repeat unit or monomer with variable size. Generally, the mean size of the monomer among satDNA families can vary between 100 and 1000 bp, although it changes widely [4]. These repeated sequences are typically found in the heterochromatin [5], being part of chromosome structures, such as telomeres [6] and pericentromeric and centromeric regions [1]. The amount of satDNA present in a eukaryotic genome depends on the organism and in insects it can be up to the 50% of the total DNA [7,8]. The pericentromeric preferential location of the repetitive DNA allows creation of a particular nuclear architecture that may determine unequal transmission or expression properties, even with the same coding sequences [9]. Because of these properties, the repetitive components in the Genes 2020, 11, 783 3 of 13 In this study, the genome of H. variegata was sequenced and bioinformatics tools were applied in order to analyze its satellitome. Its economic importance and the small genome size of H. variegata (1C = 0.29 pg) in comparison with other Coleoptera species [45] makes it very suitable for the application of genomic analysis techniques. In addition, this study may be a first step that could help in the future to obtain the complete assembled genome of this important species.

Sampling, Chromosome Preparation and DNA Extraction
Hippodamia variegata samples were provided by Biosur Insectarios (Murcia, Spain), a company focused on the production of predatory insects for biological pest control. Males were dissected and the testes were taken out for cytogenetic analysis. After dissection, the bodies were preserved in 100% ethanol at −20 • C until DNA extraction. Chromosome slides were prepared from male gonads obtained from adults [46]. Slides were stained with Giemsa, and analyzed with an Olympus (Hamburg, Germany) BX51 microscope equipped with an Olympus DP70 camera. C-banding was carried out following the protocol described by Sumner [47] including some modifications [48]. After C-banding, chromosomes were stained with DAPI (4'-6-diamino-2-fenil-indol).
Genomic DNA was isolated from adults using a commercial kit (NucleoSpin Tissue, Mini kit for DNA from cells and tissue, Macherey-Nagel Co., Düren, Germany) according to the instructions provided by the manufacturer. DNA concentration and purity were estimated by measuring the absorbance at 260 nm and 280 nm using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

DNA Sequencing and Data Analysis
For genome sequencing, approximately 4 µg of genomic DNA were used. A library of 700 bp fragments were sequenced in the Illumina ® Hiseq™ 2000 platform yielding about 3.2 Gb data of 101 bp pair-end reads. This Illumina library was deposited in the SRA database under Bioproject PRJNA644199.
A set of randomly selected 12 million pair-end reads (≈120 Mb) were used for the clustering and assembly analysis using RepeatExplorer, implemented within the Galaxy environment (www.repeatexplorer.org) [22,23]. We used default options, i.e., a minimum overlap of 55% and a similarity more than 90%. Clusters containing satDNAs were identified based on the graph topology with sphere or ring-like shapes. For each candidate cluster, we examined the contigs assembled by RepeatExplorer to search tandem repeated structures using the Dotmatcher tool (available on-line http://emboss.bioinformatics.nl/cgi-bin/emboss/dotmatcher/) and thus identify the monomer sequence for each satellite. Multiple-sequence alignments were performed using MUSCLE [49] to obtain the consensus sequences. All the obtained consensus sequences from potential satDNAs were used against Repbase using CENSOR (http://www.girinst.org/) [50]. The sequence data were also compared with the GenBank/NCBI DNA databases using the BLAST network service and the EMBL database [51]. We calculated the divergence and abundance for each satDNA using RepeatMasker [52] with the "-a" option and the RMBlast search engine. For this, we randomly selected a million reads and aligned against the total collection of satDNA dimers or monomer concatenations until approximately 200 bp in length was reached. We estimated the average divergence and generated a satellite landscape considering distances from the sequences by applying the Kimura 2-parameter model with perl script calcDivergenceFromAlign.pl and createRepeatLandscape.pl from the RepeatMasker suite.

PCR Amplification, Sequences Cloning and Cytogenetic Mapping
The consensus sequences of the most abundant satDNA families were used as a template to design a set of primers for PCR amplification (Table 1). PCR reactions were carried out in 20 µL reaction mixtures using 50 ng of genomic DNA, 0.5 mM dNTPs, 50 pmol of each primer and 1 U of Taq polymerase (Bioline, London, UK). The PCR program used was 2 min at 92 • C and 35 cycles with Genes 2020, 11, 783 4 of 13 20 s at 92 • C, 60 s at 50 • C, and 2 min at 72 • C, with a final extension of 5 min at 72 • C. After the amplification, the generated fragments were separated into 2% agarose gel electrophoresis. The expected bands were eluted from the gel using the E.Z.N.A. ® Gel Extraction kit (Omega, Norcross, GA, USA) and used for the ligation and transformation of competent Escherichia coli DH5α (Zymo Research, Orange, CA, USA) using the pGEMT-Easy vector (Promega, Southampton, UK). Recombinant plasmids were selected using LB/ampicillin/IPTG/X-Gal plates. The recombinant plasmids were purified using the E.Z.N.A. ® Plasmid Midi Kit (Omega, Norcross, GA, USA) and sequenced in both strands. Positive plasmids were labelled with biotin-16-dUTP using the Nick Translation Kit (Roche, Manheim, Germany), according to the company's instructions. These labelled plasmids were used as probes (final concentration of 5 ng/ml in 50% formamide) to perform fluorescence in situ hybridizations (FISH) according to the procedure described in Palomeque et al. [48]. The fluorescent immunological detection was carried out using the avidin-FITC/anti-avidin-biotin system with three amplification rounds. Slides were mounted in Vectashield-DAPI (Vector Laboratories, Burlingame, CA, USA). DAPI in the antifade solution was used to counterstain the chromosomes. Images were taken with a BX51 Olympus ® fluorescence microscope (Olympus, Hamburg, Germany) equipped with a CCD camera (Olympus ® DP70) and processed using Adobe ® Photoshop ® software.

Results and Discussion
Hippodamia variegata has a diploid chromosome number of 2n = 20, with a XY sexual chromosome system ( Figure 1A). Sexual chromosomes show the parachute association Xyp in meiosis I ( Figure 1B). Previously, Rozek and Holecová [53] reported the same chromosome number for populations from Central Europe. The karyotype of these specimens showed 7 pairs of subtelocentric and 2 pairs of metacentric autosomes, a metacentric X chromosome and the small Y chromosome that is dot-shaped. However, here we show that in the analyzed population, the karyotype presents three metacentric autosome pairs, the three biggest, and the metacentric X chromosome. All the remaining chromosomes seem to be subtelocentric or acrocentric, including the small Y chromosome ( Figure 1A). These differences could be due to the existence of interpopulation polymorphisms. The small size of the chromosomes does not allow one to determinate the cause of this polymorphism, which probably could be provoked by a pericentromeric inversion. Chromosome polymorphisms due to variation in chromosome number or in the chromosome morphology were earlier described in other Coccinellidae species [54,55]. The chromosome number of H. variegata is similar to that found in other Coccinellidae species, where the most common chromosome numbers are 2n = 18 or 2n = 20 [56]. The Xyp system is also the most common among Coccinellidae although sexual determination systems such as XY, X0 and neo-XY systems have also been found [56]. Genes 2020, 11, x FOR PEER REVIEW 5 of 14 Rozek and Holecová [53] reported that all the chromosomes of this species, after the C-banding, showed a big heterochromatic pericentromeric region with the exception of the Y chromosome that was completely euchromatic. However, we found that all chromosomes have relatively big pericentromeric heterochromatin and DAPI staining revealed that those heterochromatic regions are A+T rich ( Figure 1C). The main component of the heterochromatin is satellite DNA, so in order to characterize the satDNA families presented in the H. variegata, we carried out NGS of its genome.
The sequencing data of the H. variegata genome produced 32,394,082 reads corresponding to more than 3 Gb. Sequencing data shows that the A+T genome content was 63.6%. This high A+T content is a common feature within the insect genomes, and can be over 70% in some species [57]. From the total reads, a subset of 12 million pair-end reads (≈1,200 Mb) was randomly selected to analyze in RepeatExplorer. The C-value of H. variegata is 0.29 pg [58]; therefore, the estimate haploid genome size of H. variegata is about 284 Mb. RepeatExplorer recommends using genome coverages of at least ~1% [22]. We selected 500,144 sequences in the pipeline, corresponding to about 5.8% of the genome of H. variegata. After RepeatExplorer clustering, 235,066 reads were grouped into 23,169 clusters, with 47% of the H. variegata genome composed of repeated sequences ( Figure 2). All the remaining reads (265,078) were classified as singletons. From those clusters, 365 represented at least 0.001% of the genome and those that featured a star-like or circular graph topology, typically founded in the satDNA families, were deeply analyzed. After the computational analysis, we identified 29 satDNA families ( Table 2, Supplementary Table S1) (Genbank accession no. MT613047-MT613075). The satDNA families were named according to Ruiz-Ruano et al. [20], including the species name abbreviation, a number in decreasing abundance and the length of the repeat sequence, starting from HvarSat01-277 (the most abundant) to HvarSat29-105 (the least abundant). Sequences with the insect telomeric repeat TTAGG were not found through this analysis. Nevertheless, the (TTAGG)50 repeat was included in RepeatMasker analysis showing that the abundance of telomeric repeats was 0.17% (Table 2). Consequently, the H. variegata satellitome is composed of at least 30 satDNA families, corresponding to 14.93% of the genome ( Table 2). Rozek and Holecová [53] reported that all the chromosomes of this species, after the C-banding, showed a big heterochromatic pericentromeric region with the exception of the Y chromosome that was completely euchromatic. However, we found that all chromosomes have relatively big pericentromeric heterochromatin and DAPI staining revealed that those heterochromatic regions are A+T rich ( Figure 1C). The main component of the heterochromatin is satellite DNA, so in order to characterize the satDNA families presented in the H. variegata, we carried out NGS of its genome.
The sequencing data of the H. variegata genome produced 32,394,082 reads corresponding to more than 3 Gb. Sequencing data shows that the A+T genome content was 63.6%. This high A+T content is a common feature within the insect genomes, and can be over 70% in some species [57]. From the total reads, a subset of 12 million pair-end reads (≈1,200 Mb) was randomly selected to analyze in RepeatExplorer. The C-value of H. variegata is 0.29 pg [58]; therefore, the estimate haploid genome size of H. variegata is about 284 Mb. RepeatExplorer recommends using genome coverages of at least~1% [22]. We selected 500,144 sequences in the pipeline, corresponding to about 5.8% of the genome of H. variegata. After RepeatExplorer clustering, 235,066 reads were grouped into 23,169 clusters, with 47% of the H. variegata genome composed of repeated sequences ( Figure 2). All the remaining reads (265,078) were classified as singletons. From those clusters, 365 represented at least 0.001% of the genome and those that featured a star-like or circular graph topology, typically founded in the satDNA families, were deeply analyzed. After the computational analysis, we identified 29 satDNA families ( Table 2, Supplementary Table S1) (Genbank accession no. MT613047-MT613075). The satDNA families were named according to Ruiz-Ruano et al. [20], including the species name abbreviation, a number in decreasing abundance and the length of the repeat sequence, starting from HvarSat01-277 (the most abundant) to HvarSat29-105 (the least abundant). Sequences with the insect telomeric repeat TTAGG were not found through this analysis. Nevertheless, the (TTAGG) 50 repeat was included in RepeatMasker analysis showing that the abundance of telomeric repeats was 0.17% (Table 2). Consequently, the H. variegata satellitome is composed of at least 30 satDNA families, corresponding to 14.93% of the genome ( Table 2).    Using classical isolation methodologies, only one or a few satDNA families have been characterized within a species [7]. However, the application of NGS technologies has eased the characterization of a large number of satDNA families. Among insects, for example, the grasshopper Locusta migratoria, it has been possible to find the existence of 62 families of satDNA [20]. Other analyzed Orthoptera species also present numerous families of satDNA, such as Eneoptera surinamensis with 45 families [28], 11 in Gryllus assimilis [29], 10 in Ronderosia bergii [59], 27 in Eumigus monticola [60] or the amazing 76 families found in Pyrgomorpha conica [27]. Orthoptera is the group of insects with the largest genome size, with genome sizes between 1.6 and 16 Gb [45]. In other insects, such as Triatoma infestans (Hemiptera), with a genome size between 1.5 and 1.9 Gb, NGS has revealed the existence of 42 satDNAs families [17]. One of the main causes of variation in the eukaryotic genome sizes is the amount of repetitive DNAs, including satDNA. However, the genome size may not be directly related to the number of satDNA families present in it. The Coleopteran H. variegata has a substantially smaller genome size (0.28 Gb) than the species discussed above and also has a large number of satDNA families. The number of satDNA families also does not seem to be related to the total amount of repetitive DNA in the genome. In H. variegata, the 30 satDNA families, altogether, comprise 14.93% of the genome. In the fish Megaleporinus microcephalus, 164 satDNA families have been isolated, which is, by far, the most satellite-rich species discovered to date. Nevertheless, the satDNA of M. macrocephalus only represented 13.47% (female) and 11.99% (male) of the genome [61]. Therefore, the existence of multiple satDNA families seems to be a common characteristic of eukaryotic genomes as they have also been found in plants and vertebrates [61][62][63].
The H. variegata satellitome shows satDNA families that differ significantly in monomere size (Figure 3), from 5 bp (telomere) or 41 bp (HvarSat27-41), up to 2 kb (HvarSat07-2000). The most common repetitive unit size for H. variegata satDNAs is 100-200 bp. The family HvarSat07-2000, with a repeat unit of 2 kb is, to our knowledge, the largest repeat unit described so far in Coleoptera. Until now, the largest satDNA was the 1169-bp PstI family isolated in the beetle Misolampus goudoti [6]. Satellites with the largest repeat units have also been described in other insect species, for example, in the ant Monomorium subopacum, with a repeat unit of 2.5 kb [64] or a satellite with a repeat unit of 1 kb found in the kissing bug Triatoma infestans [17], although most of the satDNA isolated in insects have repeat units over 500 bp [7]. Using classical isolation methodologies, only one or a few satDNA families have been characterized within a species [7]. However, the application of NGS technologies has eased the characterization of a large number of satDNA families. Among insects, for example, the grasshopper Locusta migratoria, it has been possible to find the existence of 62 families of satDNA [20]. Other analyzed Orthoptera species also present numerous families of satDNA, such as Eneoptera surinamensis with 45 families [28], 11 in Gryllus assimilis [29], 10 in Ronderosia bergii [59], 27 in Eumigus monticola [60] or the amazing 76 families found in Pyrgomorpha conica [27]. Orthoptera is the group of insects with the largest genome size, with genome sizes between 1.6 and 16 Gb [45]. In other insects, such as Triatoma infestans (Hemiptera), with a genome size between 1.5 and 1.9 Gb, NGS has revealed the existence of 42 satDNAs families [17]. One of the main causes of variation in the eukaryotic genome sizes is the amount of repetitive DNAs, including satDNA. However, the genome size may not be directly related to the number of satDNA families present in it. The Coleopteran H. variegata has a substantially smaller genome size (0.28 Gb) than the species discussed above and also has a large number of satDNA families. The number of satDNA families also does not seem to be related to the total amount of repetitive DNA in the genome. In H. variegata, the 30 satDNA families, altogether, comprise 14.93% of the genome. In the fish Megaleporinus microcephalus, 164 satDNA families have been isolated, which is, by far, the most satellite-rich species discovered to date. Nevertheless, the satDNA of M. macrocephalus only represented 13.47% (female) and 11.99% (male) of the genome [61]. Therefore, the existence of multiple satDNA families seems to be a common characteristic of eukaryotic genomes as they have also been found in plants and vertebrates [61][62][63].
The H. variegata satellitome shows satDNA families that differ significantly in monomere size (Figure 3), from 5 bp (telomere) or 41 bp (HvarSat27-41), up to 2 kb (HvarSat07-2000). The most common repetitive unit size for H. variegata satDNAs is 100-200 bp. The family HvarSat07-2000, with a repeat unit of 2 kb is, to our knowledge, the largest repeat unit described so far in Coleoptera. Until now, the largest satDNA was the 1169-bp PstI family isolated in the beetle Misolampus goudoti [6]. Satellites with the largest repeat units have also been described in other insect species, for example, in the ant Monomorium subopacum, with a repeat unit of 2.5 kb [64] or a satellite with a repeat unit of 1 kb found in the kissing bug Triatoma infestans [17], although most of the satDNA isolated in insects have repeat units over 500 bp [7].  The A+T content in the satDNA families of H. variegata ranges between 45.6 and 74.7% (Table 2). Traditionally it has been assumed that satellite DNA is rich in A+T, since most have an A+T content over 50% [7]. The A+T richness of the H. variegata genome is 63.6%. The number of satDNA families with A+T content above and below this value are equally distributed (Table 2). Therefore, there does not seem to be any tendency towards the enrichment of satellite DNA in A+T in relation to the complete genome. The HvarSat01-277 family is the main satDNA family in H. variegata and represents 9.37% of the genome ( Table 2). The second most abundant family (HvarSat02-127) represents only 2% of the genome, with the remaining families below 1%. In fact, most satDNA families (21 out of 30) are below 0.1%. The existence of a main satDNA family, clearly the most abundant one, is a common characteristic of insect genomes [7]. However, there are species without a clear main satDNA family and different families of satDNA are located on the heterochromatic regions [20,28,29].
Divergence of the H. variegata satellitome, estimated with RepeatMasker [52], is lower than in others insects in which the same methodology has been used [20,27,63]. The divergence among satDNA families ranges between 0.23% and 25.74%, with a median value being 5.75% ( Table 2).
The minimum values are shown by the telomeric repeat (0.23%) and the HvarSat07-2000 family (1.31%), whereas the highest value is shown by HvarSat16-87 (25.75%). Divergence of satDNA is inversely related to amplification and homogenization processes and directly related to mutation rate [62,65]. Consequently, low divergence in H. variegata satDNA families may reflect a tendency for homogenizations, which is also reflected in the satellitome landscape representing abundance (y-axis) and divergence (x-axis) for each satDNA with respect to each consensus sequence (Figure 4, Supplementary Figure S1). The main peak on the distribution shows that most satDNA sequences show divergence below 5%. That figure is shared for each satDNA family in separate repeat landscapes (Supplementary Figure S1). Interestingly, HvarSat01-277, the most abundant satDNA in H. variegata, shows two peaks corresponding with two types of repeat units differing in divergence (Supplementary Figure S3). The double peak pattern in a repeat landscape is not unusual, for example, in the fern Vandenboschia speciosa VspSat01-59, where telomeric repeats present two peaks on their repeat landscape [62].
Genes 2020, 11, x FOR PEER REVIEW 8 of 14 The A+T content in the satDNA families of H. variegata ranges between 45.6 and 74.7% (Table 2). Traditionally it has been assumed that satellite DNA is rich in A+T, since most have an A+T content over 50% [7]. The A+T richness of the H. variegata genome is 63.6%. The number of satDNA families with A+T content above and below this value are equally distributed (Table 2). Therefore, there does not seem to be any tendency towards the enrichment of satellite DNA in A+T in relation to the complete genome. The HvarSat01-277 family is the main satDNA family in H. variegata and represents 9.37% of the genome ( Table 2). The second most abundant family (HvarSat02-127) represents only 2% of the genome, with the remaining families below 1%. In fact, most satDNA families (21 out of 30) are below 0.1%. The existence of a main satDNA family, clearly the most abundant one, is a common characteristic of insect genomes [7]. However, there are species without a clear main satDNA family and different families of satDNA are located on the heterochromatic regions [20,28,29].
Divergence of the H. variegata satellitome, estimated with RepeatMasker [52], is lower than in others insects in which the same methodology has been used [20,27,63]. The divergence among satDNA families ranges between 0.23% and 25.74%, with a median value being 5.75% ( Table 2). The minimum values are shown by the telomeric repeat (0.23%) and the HvarSat07-2000 family (1.31%), whereas the highest value is shown by HvarSat16-87 (25.75%). Divergence of satDNA is inversely related to amplification and homogenization processes and directly related to mutation rate [62,65]. Consequently, low divergence in H. variegata satDNA families may reflect a tendency for homogenizations, which is also reflected in the satellitome landscape representing abundance (y-axis) and divergence (x-axis) for each satDNA with respect to each consensus sequence (Figure 4, Supplementary Figure S1). The main peak on the distribution shows that most satDNA sequences show divergence below 5%. That figure is shared for each satDNA family in separate repeat landscapes (Supplementary Figure S1). Interestingly, HvarSat01-277, the most abundant satDNA in H. variegata, shows two peaks corresponding with two types of repeat units differing in divergence (Supplementary Figure S3). The double peak pattern in a repeat landscape is not unusual, for example, in the fern Vandenboschia speciosa VspSat01-59, where telomeric repeats present two peaks on their repeat landscape [62]. Searches in Repbase using CENSOR (http://www.girinst.org/) failed to find sequences with significant similarity with the H. variegata satDNA families. Nucleotide sequences of the H. variegata satDNA families were also compared with the GenBank/NCBI DNA databases, obtaining only positive results for the HvarSat16-87 family. Sequences with similarity with the consensus sequence of HvarSat16-87 were found in two scaffolds obtained from the assembled genome of another Coccinellidae species, Harmonia axyridis [66]. These two scaffolds (accession no. AP018897, AP018898) contain five tandem arrays of repeated sequences (Supplementary Figure S2). From each Searches in Repbase using CENSOR (http://www.girinst.org/) failed to find sequences with significant similarity with the H. variegata satDNA families. Nucleotide sequences of the H. variegata satDNA families were also compared with the GenBank/NCBI DNA databases, obtaining only positive results for the HvarSat16-87 family. Sequences with similarity with the consensus sequence of HvarSat16-87 were found in two scaffolds obtained from the assembled genome of another Coccinellidae species, Harmonia axyridis [66]. These two scaffolds (accession no. AP018897, AP018898) contain five tandem arrays of repeated sequences (Supplementary Figure S2). From each tandem array we obtained monomers and generated a consensus sequence of 87 bp in length and 74.71% similarity with the consensus sequence of the HvarSat16-87 family. Interestingly, the HvarSat16-87 family is the most variable satDNA in the H. variegata genome (25.74% of divergence) and a similar variability was found for this repeat sequence in Harmonia axyridis (24.30% of divergence). These results are consistent with phylogenetic studies that show that Harmonia is the sister-genus of the genus Hippodamia [67,68].
PCR amplifications for FISH probe generation were performed for the four most abundant satDNA families. Hybridization with the HvarSat01-277 probe shows a pericentromeric location on the chromosomes, coincident with the location of the heterochromatin revealed by C-banding and DAPI staining ( Figure 5A,B). This result coincides with that observed in most satDNA families isolated from beetles, as they are often located in heterochromatic chromosomal regions rich in A+T. The most abundant satDNA is usually the main component of the heterochromatin located in the pericentromeric regions [4,14]. In addition, pericentromeric satDNA has an essential role in the formation and maintenance of kinetochorus, as well as in chromosome segregation and sister chromatid cohesion because it is involved in the stabilization of DNA-binding proteins [69][70][71]. Funding: This work was supported by the Universidad de Jaén (through the program "Plan de Apoyo a la Hybridization signals for the HvarSat02-127 family were located only on two small bivalents and the X chromosome ( Figure 5C,D), whereas HvarSat03-217 signals were located on one bivalent only ( Figure 5E,F). Due to their restricted location, these satDNAs could be very useful as chromosome markers, allowing one to distinguish those chromosomes pairs as well as the X chromosome from other chromosomes. No clear hybridization signals were obtained with the HvarSat04-487 satDNA probe. It is possible that this satDNA has a dispersed location in the H. variegata genome and therefore does not generate visible signs of hybridization. The existence of short arrays of satDNA along the chromosomes could result in the absence of hybridization signals or the presence of a bright background [20,27]. Traditionally, satDNA has been considered as DNA located on the heterochromatic regions, but euchromatin also possesses tandem repeats as microsatellites, minisatellites, and even satDNA [17,20,25,72].
In spite of their ecological and economic importance, the data about repetitive DNA in Coccinellidae are scarce as we indicated previously. H. variegata is the first Coccinellidae beetle whose satellitome has been studied by applying new sequencing technologies and bioinformatics tools. Recently, Louzada et al. [73] carried out a review of the important role that satellite DNA, along with other types of repetitive DNA, plays in the architecture and evolution of the genome. These authors highlight the importance of the application of the new technologies in the study of these sequences.
Nowadays, knowing the composition and structure of genomes is a key not only for understanding their past evolution but also for discerning their future perspective. The H. variagata satellitome has shed some light about genome composition and structure on this species. Furthermore, it would be helpful for mapping scaffolds to chromosomes in a future genome assembly project, leading to a complete and well-annotated genome. Funding: This work was supported by the Universidad de Jaén (through the program "Plan de Apoyo a la Investigación 2019-2020", Acción 1).

Conflicts of Interest:
The authors declare no conflict of interest.