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Satellitome represents the collection of different satellite DNAs (satDNAs) in a genome. SatDNAs are tandemly repeated non-coding DNA sequences that constitute the most abundant and the fastest evolving part of the eukaryotic genome. Due to their extreme diversity and methodological difficulties to characterize and compare satDNA collection in complex genomes, knowledge on their putative functional constraints and capacity to participate in genome evolution remains rather elusive. SatDNA transcripts have been detected in many species. New studies have shown that deregulation of satDNA expression has role in cancer progression. However, how satDNA transcripts are regulated on the genome-wide scale is poorly understood. Here, we conducted a genome-wide survey and comparative analyses of satelitomes among different closely related Meloidogyne nematodes. The evolutionary trends of satDNA on the genome- wide scale show that each round of proposed polyploidization in the evolutionary history of the investigated species is concomitant with the addition of a new subset of satDNAs in the satellitome of any particular Meloidogyne species. Successive incorporation of new sets of satDNAs in the genome along the process of polyploidization suggests multiple hybridization events as the main factor in the formation of these species. In addition, comparative expression analyses of complete satDNA set show similar transcription profile of satDNAs among different Meloidogyne species which is not dependent on satDNA genome abundance indicating similar mechanisms that operate in regulation of satDNA expression in related genomes. Furthermore, stage-specific expression pattern of satDNAs through the Meloidogyne incognita development supports the view that satDNA expression is not a stochastic event but is subject to coordinated control. Comparative analyses of 83 satDNA sequences detected in Meloidogyne satelitomes revealed conserved sequence features indicative of evolution under selective pressure and their putative functional potential. Our results demonstrate the power of comparative analysis of the non-coding genome part for the successful elucidation of the origin of species with a complex history. Whereas satDNAs generally evolve extremely quickly and their functional role is largely difficult to be proved, comparative analyses of satellitomes and satDNA transcriptomes in related genomes could be a way to address satDNA regulation and their putative functional potential.

Additional file 1: Fig. S1. Different genome coverages (0,125X, 0,25X and 0,5X) applied in graph-based clustering analysis by Repeat Explorer with number of total obtained clusters on isolates used for comparative analysis; M. incognita L19 (SRR4242479), M. javanica VW4 (SRR4242459), equal parts of three isolates (HarA, L32, L28) for M. arenaria (SRR4242477, SRR4242480, SRR4242481) and M. floridensis SJF1 (SRR4242475). Fig. S2. Comparative analysis of satellitomes in three different isolates of (A) M. incognita (isolates 56, 61, 79), (B) M. javanica (isolates 59, 71, 78), and (C) M. arenaria (isolates 77, 80, 81). Each column represents putative satDNA, while the areas of the colored rectangles are proportional to the abundance of individual satDNA in the genome ranging from 0-0.594% in M. incognita, 0-0.649% in M. javanica and 0-0.694% in M arenaria. Fig. S3. Comparison of MelSat transcripts of the two different transcriptome data sets from M. incognita using the Bowtie2 and BBMap mapper. Fig. S4. Alignments of six MelSat groups which show significant mutual repeat unit similarity (A-F). MelSat60, MelSat65 and MelSat76 (E) represent previously published MARJA, MPA1 and AJL satDNAs respectively [12, 38, 39]. MV1, LV1, MV2, LV2 and HV indicate domains of MEL 172 satDNA described previously [12]. CENP-B box-like sequence (F) previously found in distant M. chitwoodi and M. fallax [10]. Fig. S5. (A) Identity matrix of CENP-B box containing satDNAs (MelSat 72/02/36/42/61/83 from MIG species and from M. chitwoodi and M. fallax (ChFa) previous published in [10]) and (B) identity matrix of MelSat01 in different Meloidogyne species (Minc-M. incognita, Mflo-M. floridensis, Mare-M. arenaria, Mjav-M. javanica, Ment-M. enterolobii and Mhap-M. haplanaria). Fig. S6. Electrophoresis of FISH probes after PCR labeling and purification; (A) biotin-labeled probe for MelSat01 (spliced leader) amplified from a cloned dimer [33] (B) six MelSat probes (CENP-B box containing) amplified and labeled with Cy3 from M. arenaria genomic DNA (MelSat02, 36, 61, 72, 83) and from a cloned dimer of MelSat42. Table S1. Main characteristics of the 83 satDNAs found in the genomes of Meloidogyne species by RepeatExplorer based on consensus sequences of satDNA family found in each of the analyzed species. The slash symbol in the divergency column indicates that this satDNA family is only present in one species. Table S2. Statistics of satDNAs from M. incognita satellitome mapped on genome assembly [42] and unplaced reads. Table S3. Satellite DNA transcription data of Meloidogyne species. SatDNA transcription was obtained with Bowtie2 mapping and normalization using RPKM (reads per kilobase of transcript per million mapped reads) method. SatDNA are ordered based on their catalog number as shown in Fig. 2. Other details are described in Methods section Analysis of the whole transcriptome data. Table S4. Expression analyses of 4 house-keeping genes in M. incognita based on previously validated candidates [65]. For mapping of two juvenile (J2) transcriptome databases (ERR790027 and ERR790028), coding sequences (CDS) of reference genes downloaded from WormBase ParaSite ( https://parasite.wormbase.org/index.html ) were used as listed M. incognita loci for each gene. Table S5. Consensus sequences of 83 satDNAs found in the genomes of the Meloidogyne species by RepeatExplorer. Table S6. Primers pairs used for MelSat amplification and labeling.

Bivalve molluscs are a large group of economically and ecologically important invertebrates, which made them particularly interesting as model organisms for genomic research. So far, 31 bivalve genomes have been sequenced, the first one corresponding to the Pacific oyster (Crassostrea gigas). Segments of the genome enriched in repetitive sequences still present a challenge in the genome assemblies, leaving the composition of DNA sequences underlying the heterochromatic regions and the centromeres still unexplored in many organisms. The typical landmark of centromere chromatin is a variant of the H3 histone, CenH3, while DNA sequences building constitutive heterochromatin are associated with H3K9me3- histone modification. In order to investigate the DNA composition of genomic regions enriched in repetitive sequences, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the whole genome and CenH3- and H3K9me3-associated sequences. The obtained sequences were analysed using RepeatExplorer pipeline. Fifty-two satellite DNAs constitute the satellitome. CenH3-associated sequences resulted in diverse repetitive DNA content and were assigned to six different groups of repetitive sequences. Several sequences enriched in CenH3-ChIP were localized on metaphase chromosomes using fluorescence in situ hybridization, each encompassing centromeres of only a few chromosomes of C. gigas. The same sequences were also colocalized with CenH3 on male gonadal cells in different stages of spermatogenesis, revealing stage-specific distribution of the CenH3. The heterochromatin of C. gigas exhibited low abundance and localization limited on two chromosomal pairs, with H3K9me3- associated sequences being dominantly composed of DNA transposons. To conclude, we identified, characterized and localized the most abundant repetitive sequences present in the genome of the Pacific oyster.

Centromeres are chromosomal regions essential for kinetochore attachment to mitotic spindle and proper chromosome segregation during cell divisions. They are determined epigenetically with centromere-specific variant of histone H3 (CenH3). While centromere function is conserved, CenH3 and underlying DNA evolve rapidly. Most research has been done on sexual species with monocentric chromosomes characterized by a single CenH3-containing region. In contrast, holocentromeres have their function dispersed throughout the entire length of chromosome. In this study, we used holocentric nematodes of the genus Meloidogyne as a model organism for the centromere study [1]. CenH3 characterization is done for three closely related, polyploid mitotic parthenogenetic species together with distantly related meiotic parthenogen species. We found that ancestral duplication of CenH3 gene resulted in one conserved CenH3 (αCenH3) and other gene that evolved rapidly into four different CenH3 variants. Immunofluorescence performed on mitotic M. incognita revealed a dominant role of αCenH3 histone on its centromere (Figure 1) while the other CenH3s have lost their function. In addition, ChIP-Seq analysis showed that αCenH3-associated DNA is comprised of tandem repeats with divergent monomers but completely conserved 19-bp long box. Preservation of αCenH3 and DNA constituents is also proven for other investigated species. These results elucidate unique characteristics where CenH3 and associated DNA are maintained over long period of time in holocentromeres of exclusively mitotic species. Our findings of centromere evolution in asexual, holocentric organisms thus help building an integrated view on the centromere (epi)genomics.

Školjkaši su ekonomski važan razred beskralješnjaka, osobito u marikulturi. Osim ekonomskog značaja, oni su nezamjenjivi dio ekosustava u kojem sudjeluju u prijenosu organskih tvari i minerala. Stoga se broj analiziranih genoma školjkaša višestruko povećao u zadnjih nekoliko godina. Trenutno je sekvencirano 20 školjkaških genoma. Prvi sekvencirani je genom pacifičke kamenice (Crassostrea gigas) koji je parcijalno sastavljen do razine genomskih odsječaka (engl. scaffold). Veličina genoma pacifičke kamenice procjenjuje se na 558 Mb, a osobit je po tome što sadrži mali udio heterokromatina i mali udio satelitnih DNA. Centromere su kromosomski lokusi neophodni za pravilno razdvajanje genetskog materijala tijekom stanične diobe. Iako su centromere funkcionalno stabilne i strukturno visokoočuvane, centromerna DNA mijenja se brzo tijekom evolucije, a zajedno s njom i specifični proteini koji se vežu za centromernu DNA. Jedan od specifično vezanih proteina jest centromerna varijanta histona H3 koji u centromernim nukleosomima zamjenjuje kanonski H3 te predstavlja epigenetičku determinantu centromere. Centromerna i pericentromerna područja DNA često u svom sastavu imaju ponovljene sekvence. Kako bismo identificirali sekvence DNA koje izgrađuju centromerne lokuse na kromosomima pacifičke kamenice, napravili smo kromatinsku imunoprecipitaciju s antitijelom specifičnim za centromerni histon H3 ove vrste. Sekvenciranjem nove generacije odredili smo nukleotidni slijed imunoprecipitiranih segmenata DNA, kao i cjelokupne genomske DNA. Dobivene sekvence bionformatički su analizirane. Ponovljene sekvence DNA za koje se pokazalo da su obogaćene u centromernoj frakciji lokalizirane su metodom fluorescenscijske hibridizacije in situ (FISH) na metafaznim kromosomima. Ponovljene sekvence centromernih kandidata i protein CenH3 imunofluorescenscijski su kolokalizirani na gonadnim stanicama u različitim fazama spermatogeneze.

The flour beetles Tribolium castaneum and Tribolium freemani, the important world-wide pests of stored agricultural products, are able to mate, but produce sterile hybrid progeny in reciprocal crosses. In the context of genetic diversity between closely related species, we focused our research on DNA and protein aspects of their centromere regions as crucial chromosomal structures. By using the histone H3 sequence as query to search genome databases, in both species we first identified the centromeric histone H3 variants (CENH3), epigenetic hallmarks of active centromeres. T. castaneum cCENH3 and T. freemani fCENH3 H3-like variants share 80% identity in amino acid sequences. Their species-specific N-terminal tails were used to raise antibodies that localized cCENH3 and fCENH3 proteins exclusively to centromeres of T. castaneum and T. freemani, respectively. We further performed ChIP-Seq experiments, which predominantly associated cCENH3 and fCENH3 nucleosomes with a major satellite DNA in each species. As supported by IF-FISH analyses, highly abundant satellite DNAs, TCAST in T. castaneum and TFREE in T. freemani, spread both at pericentromeric and functional centromeric regions of all chromosomes. TCAST and TFREE satellite DNAs account for large fractions (>20%) of their indigenous genomes, and they differ dramatically in monomer unit length (360 bp versus 166 bp) as well as in nucleotide sequence, thus being fundamentally species- specific. In terms of the reproductive isolation between T. castaneum and T. freemani, these findings argue in favor of adaptive evolution of cCENH3 and fCENH3 proteins that might be driven by a response to changes in satellite DNA sequences at centromere loci.

Arrays of DNA sequences repeated in tandem, commonly known as satellite DNAs (satDNAs), build heterochromatic chromosomal compartments of almost every eukaryotic species. Many satDNAs differing in sequence, copy number, and chromosomal localization populate a genome. Non- coding character, repetitiveness, difficulties in cloning, sequencing and assembly of hundreds and thousands of nearly-identical monomers made these sequences poorly explored, particularly regarding their possible roles in genome function and evolution. Tandem repeats are a preferred form of DNA sequences in pericentromeres, spanning also the functional centromere, a domain responsible for proper segregation of chromosomes in cell divisions. However, there is no particular centromeric DNA, and centromeres are epigenetically determined by histone H3 protein variant CENH3. CENH3 is also a species-specific marker of centromere function, owing to the association of its rapidly evolving N-terminal part with underlying DNA sequences. According to the library model, species-specific differences in satDNA composition are explained as a result of copy number alterations within a set, regularly shared by a group of related species. In addition, evolution of satDNA nucleotide sequences can be surprisingly slow, and some can persist for extremely long evolutionary periods. Features of satDNA sequence dynamics might be advantageous for maintaining DNA-protein interactions in the centromere, allowing in the same time rapid replacements and stability of satDNA variants. To keep functionality, both DNA and protein components must evolve in accord, and they drive each other’s evolution. Once accumulated in groups of individuals, changes in the centromeric satDNA and in the corresponding CENH3 can cause incompatibilities in chromosome pairing in hybrids, thus triggering reproductive isolation and speciation. Recent advances in NGS sequencing and in bioinformatic tools enabled characterization of a whole-genome complement of satellite DNAs, the satellitome. In combination with chromatin immunoprecipitation using CENH3- specific antibodies, it is now possible to focus onto details of composition of DNA sequences within and around centromeres of a variety of experimental organisms.

Centromeres are chromosomal domains essential for proper segregation of genetic material in cell divisions. They are defined epigenetically by the presence of centromere-specific histone H3 protein (CENH3). CENH3 histone is a species-specific protein, particularly variable in its N-terminal domain which is associated with centromeric DNA. In contrast to conserved function of the centromere, CENH3 and centromeric DNA evolve rapidly in diverse organisms. Why the centromere is composed of unstable units is still unsolved question. Nematodes Meloidogyne have some exceptional characteristics, such as presumptive holocentric organization of the centromere and specific mode of reproduction by mitotic parthenogenesis, which make them an ideal system to study centromere structure and function. Interestingly, our genome analysis of centromere protein CENH3 in Meloidogyne incognita reveal three divergent genes for CENH3 (α, β, λ), which are completely conserved in its related species. Transcriptome data show developmental stage-specific expression of CENH3 variants. Immunostaining of chromosomes with antibodies specific for αCENH3 reveals so far unseen distribution of CENH3 in forms of monocentric, dicentric, polycentric and holocentric centromeres. ChIP experiments suggested that different repetitive sequences with conserved 21-nt motif may be associated with αCENH3 domains.

Centromeres are essential chromosomal loci responsible for proper chromosome segregation during cell division. Centromeric chromatin is epigenetically defined by centromere-specific histone H3 variants, CENH3 proteins, that show significant heterogeneity among eukaryotic organisms. Concurrently, underlying DNA sequences, mostly composed of satellite DNAs (satDNAs) and transposons, are highly variable even between closely related species. To deepen understanding of centromere evolution, we focused our research on the three insect species from the genus Tribolium: the sibling species Tribolium castaneum and Tribolium freemani, capable of mutual mating, and phylogenetically more distant Tribolium madens. Relying on 40% similarity to the histone H3 amino acid sequence, we identified the CENH3 genes in all three species. Consistently with their phylogenetic relationships, T. castaneum cCENH3 and T. freemani fCENH3 proteins share 80% identity in amino acid sequence, while both of them are less similar to T. madens mCENH3 showing 74% identity. Based on their species-specific N- terminal sequences, we raised antibodies that detected cCENH3, fCENH3, and mCENH3 exclusively in the centromeres of T. castaneum, T. freemani, and T. madens, respectively. To disclose the composition of Tribolium centromeric DNAs, we performed ChIP-Seq experiments. Illumina sequencing revealed that the cCENH3, fCENH3, and mCENH3 nucleosomes predominantly associate with the major satDNAs of each species. IF-FISH experiments confirmed that in the three examined species their highly abundant, major satDNAs span across the functional centromeres of all chromosomes. Showing no similarity in nucleotide sequence, the major satDNAs of the three species are considered species-specific. It could be hypothesized that dramatic changes in composition of centromeric DNA sequences foster adaptive evolution of CENH3 proteins. Such premise is in accordance with the centromere paradox, which conciliates the rapidly evolving centromeric DNA and CENH3 proteins conflicting conserved centromere function.