Long-read genome assembly of the insect model organism reveals spread of satellite DNA in gene-rich regions by recurrent burst events.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences that are mostly underrepresented in genome assemblies. Here we combined nanopore long-read sequencing with a reference-guided assembly approach to generate an improved, high-quality genome assembly, TcasONT, of the model beetle Enriched by 45 Mb in repetitive regions, the new assembly comprises almost the entire genome sequence. We use the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs.

The Low-Copy-Number Satellite DNAs of the Model Beetle .

The red flour beetle is an important pest of stored agricultural products and the first beetle whose genome was sequenced. So far, one high-copy-number and ten moderate-copy-number satellite DNAs (satDNAs) have been described in the assembled part of its genome. In this work, we aimed to catalog the entire collection of satDNAs.

Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs.

Background: Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences that belong to the most abundant and the fastest evolving parts of the eukaryotic genome. A satellitome represents the collection of different satDNAs in a genome. Due to 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.

Reference-Guided De Novo Genome Assembly of the Flour Beetle .

The flour beetle is a sibling species of the model organism and important pest . The two species are so closely related that they can produce hybrid progeny, but the genetic basis of their differences has not been revealed. In this work, we sequenced the genome by applying PacBio HiFi technology.

Isolation of High Molecular Weight DNA from the Model Beetle for Nanopore Sequencing.

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons.

CenH3 distribution reveals extended centromeres in the model beetle Tribolium castaneum.

Centromeres are chromosomal domains essential for kinetochore assembly and correct chromosome segregation. Inconsistent in their underlying DNA sequences, centromeres are defined epigenetically by the presence of the centromere-specific histone H3 variant CenH3. Most of the analyzed eukaryotes have monocentric chromosomes in which CenH3 proteins deposit into a single, primary constriction visible at metaphase chromosomes.

Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae).

Tandemly repeated DNAs usually constitute significant portions of eukaryotic genomes. In bivalves, however, repetitive DNAs are habitually not widespread. In our search for abundant repetitive DNAs in trough shells, we discovered a novel satellite DNA, SSUsat, which constitutes at least 1.

Structural and functional liaisons between transposable elements and satellite DNAs.

Transposable elements (TEs) and satellite DNAs (satDNAs) are typically identified as major repetitive DNA components in eukaryotic genomes. TEs are DNA segments able to move throughout a genome while satDNAs are tandemly repeated sequences organized in long arrays. Both classes of repetitive sequences are extremely diverse, and many TEs and satDNAs exist within a genome.

Centromere identity from the DNA point of view.

The centromere is a chromosomal locus responsible for the faithful segregation of genetic material during cell division. It has become evident that centromeres can be established literally on any DNA sequence, and the possible synergy between DNA sequences and the most prominent centromere identifiers, protein components, and epigenetic marks remains uncertain. However, some evolutionary preferences seem to exist, and long-term established centromeres are frequently formed on long arrays of satellite DNAs and/or transposable elements.

TCAGG, an alternative telomeric sequence in insects.

The TTAGG repeat, the only determined telomerase-dependent sequence in the Insecta, is generally reputed to be the canonical telomeric motif within the class. By studying the distribution of telomeric DNAs in 30 coleopteran beetles using Southern hybridization, BAL 31 DNA end-degradation assay and fluorescence in situ hybridization, we showed that arrays built of a TCAGG repeat substitute for (TTAGG)n sequences in all tested species within the superfamily Tenebrionoidea. We also provided the experimental evidence that (TCAGG)n repeats represent the terminal sequences on all chromosomes of the model species Tribolium castaneum.

Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells.

Human centromeres contain multi-megabase-sized arrays of alpha satellite DNA, a family of satellite DNA repeats based on a tandemly arranged 171 bp monomer. The centromere-specific histone protein CENP-A is assembled on alpha satellite DNA within the primary constriction, but does not extend along its entire length. CENP-A domains have been estimated to extend over 2,500 kb of alpha satellite DNA.

Parallelism in evolution of highly repetitive DNAs in sibling species.

Characterization of heterochromatin in the flour beetle Tribolium audax revealed two highly repetitive DNA families, named TAUD1 and TAUD2, which together constitute almost 60% of the whole genome. Both families originated from a common ancestral approximately 110-bp repeating unit. Tandem arrangement of these elements in TAUD1 is typical for satellite DNAs, whereas TAUD2 represents a dispersed family based on 1412-bp complex higher-order repeats composed of inversely oriented approximately 110 bp units.

Histone modifications within the human X centromere region.

Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus.

Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing.

The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing.

Satellite DNA junctions identify the potential origin of new repetitive elements in the beetle Tribolium madens.

Two related satellite DNA families (satellite I and satellite II) with complex higher-order repeat (HOR) monomers represent major DNA components equilocated in the pericentromeric heterochromatin of all Tribolium madens chromosomes. Fragments obtained upon genomic DNA restriction revealed two subfamilies of satellite II monomers, and also identified regions of transition between satellite I and satellite II sequences. The two subfamilies differ not only in diagnostic nucleotides, but also in flipped orientation of constituent subunits.

Preservation and high sequence conservation of satellite DNAs suggest functional constraints.

Due to a high evolutionary turnover many satellite DNAs are restricted to a group of closely related species. Here we demonstrate that the satellite DNA family PSUB, abundant in the beetle Palorus subdepressus, is distributed in a low number of copies among diverse taxa of Coleoptera (Insecta), some of them separated for an evolutionary period of up to 60 Myr. Comparison of PSUB cloned from the species Tribolium brevicornis with the PSUB family previously characterized in Palorus subdepressus revealed high sequence conservation and absence of fixed species-specific mutations.

Long inversely oriented subunits form a complex monomer of Tribolium brevicornis satellite DNA.

Highly abundant satellite DNA named TBREV is detected and characterized in the beetle Tribolium brevicornis (Insecta: Coleoptera). An outstanding peculiarity of the TBREV satellite monomer is its complex structure based on the two approximately 470-bp-long subunits, inversely oriented within a 1061-bp-long monomer sequence. The proposed evolutionary history demonstrates a clear trend toward increased complexity and length of the TBREV satellite monomer.

Conserved patterns in the evolution of Tribolium satellite DNAs.

Two satellite DNAs, TANAPH and TDEST, isolated from the beetle species Tribolium anaphe and Tribolium destructor, respectively, are characterized and compared with previously described Tribolium satellites, in order to deduce possible constraints on satellite sequence evolution between closely related species. Sequence diversity analysis of cloned monomers reveals the presence of variable and conserved segments in both satellites. In addition, non-random organization of As or Ts and their periodical distribution in the form of A or T >/=3 tracts, as well as CENP-B box-like motifs and dyad structures have been found in both satellites.

Sequence of PRAT satellite DNA "frozen" in some Coleopteran species.

The intriguing diversity of highly abundant satellite repeats found even among closely related species can result from processes leading to dramatic changes in copy number of a particular sequence in the genome and not from rapid accumulation of mutations. To test this hypothesis, we investigated the distribution of the PRAT satellite DNA family, a highly abundant major satellite in the coleopteran species Palorus ratzeburgii, in eight species belonging to the related genera ( Tribolium, Tenebrio, Latheticus), the subfamily (Pimeliinae), and the family (Chrysomelidae). Dot blot analysis and PCR assay followed by Southern hybridization revealed that the PRAT satellite, in the form of low-copy number repeats, was present in all tested species.