Repeat Elements Research Articles

A High-Quality Genome Assembly of Striped Catfish (Pangasianodon hypophthalmus) Based on Highly Accurate Long-Read HiFi Sequencing Data.

The HiFi sequencing technology yields highly accurate long-read data with accuracies greater than 99.9% that can be used to improve results for complex applications such as genome assembly. Our study presents a high-quality chromosome-scale genome assembly of striped catfish (Pangasianodon hypophthalmus), a commercially important species cultured mainly in Vietnam, integrating HiFi reads and Hi-C data. A 788.4 Mb genome containing 381 scaffolds with an N50 length of 21.8 Mb has been obtained from HiFi reads. These scaffolds have been further ordered and clustered into 30 chromosome groups, ranging from 1.4 to 57.6 Mb, based on Hi-C data. The present updated assembly has a contig N50 of 14.7 Mb, representing a 245-fold and 4.2-fold improvement over the previous Illumina and Illumina-Nanopore-Hi-C based version, respectively. In addition, the proportion of repeat elements and BUSCO genes identified in our genome is remarkably higher than in the two previously released striped catfish genomes. These results highlight the power of using HiFi reads to assemble the highly repetitive regions and to improve the quality of genome assembly. The updated, high-quality genome assembled in this work will provide a valuable genomic resource for future population genetics, conservation biology and selective breeding studies of striped catfish.

An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations

Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although the contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map an ∼10-kb microtia locus (odds ratio = 4.7; P = 6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526 to 78555137). While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (<1%). We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals. The locus is located near a chromatin loop region that regulates ROBO1 and ROBO2 expression in induced pluripotent stem cell–derived neural crest cells. Furthermore, we use single nuclear RNA sequencing to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and is shared by some East Asian subjects with craniofacial malformations, we propose that both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia.

Genomic Survey and Resources for the Boring Giant Clam Tridacna crocea.

The boring giant clam Tridacna crocea is an evolutionary, ecologically, economically, and culturally important reef-dwelling bivalve targeted by a profitable ornamental fishery in the Indo-Pacific Ocean. In this study, we developed genomic resources for T. crocea. Using low-pass (=low-coverage, ~6×) short read sequencing, this study, for the first time, estimated the genome size, unique genome content, and nuclear repetitive elements, including the 45S rRNA DNA operon, in T. crocea. Furthermore, we tested if the mitochondrial genome can be assembled from RNA sequencing data. The haploid genome size estimated using a k-mer strategy was 1.31–1.39 Gbp, which is well within the range reported before for other members of the family Cardiidae. Unique genome content estimates using different k-mers indicated that nearly a third and probably at least 50% of the genome of T. crocea was composed of repetitive elements. A large portion of repetitive sequences could not be assigned to known repeat element families. Taking into consideration only annotated repetitive elements, the most common were classified as Satellite DNA which were more common than Class I-LINE and Class I-LTR Ty3-gypsy retrotransposon elements. The nuclear ribosomal operon in T. crocea was partially assembled into two contigs, one encoding the complete ssrDNA and 5.8S rDNA unit and a second comprising a partial lsrDNA. A nearly complete mitochondrial genome (92%) was assembled from RNA-seq. These newly developed genomic resources are highly relevant for improving our understanding of the biology of T. crocea and for the development of conservation plans and the fisheries management of this iconic reef-dwelling invertebrate.

A haploid pseudo-chromosome genome assembly for a keystone sagebrush species of western North American rangelands.

Increased ecological disturbances, species invasions, and climate change are creating severe conservation problems for several plant species that are widespread and foundational. Understanding the genetic diversity of these species and how it relates to adaptation to these stressors are necessary for guiding conservation and restoration efforts. This need is particularly acute for big sagebrush (Artemisia tridentata; Asteraceae), which was once the dominant shrub over 1,000,000 km2 in western North America but has since retracted by half and thus has become the target of one of the largest restoration seeding efforts globally. Here, we present the first reference-quality genome assembly for an ecologically important subspecies of big sagebrush (A. tridentata subsp. tridentata) based on short and long reads, as well as chromatin proximity ligation data analyzed using the HiRise pipeline. The final 4.2-Gb assembly consists of 5,492 scaffolds, with nine pseudo-chromosomal scaffolds (nine scaffolds comprising at least 90% of the assembled genome; n = 9). The assembly contains an estimated 43,377 genes based on ab initio gene discovery and transcriptional data analyzed using the MAKER pipeline, with 91.37% of BUSCOs being completely assembled. The final assembly was highly repetitive, with repeat elements comprising 77.99% of the genome, making the Artemisia tridentata subsp. tridentata genome one of the most highly repetitive plant genomes to be sequenced and assembled. This genome assembly advances studies on plant adaptation to drought and heat stress and provides a valuable tool for future genomic research.

Integration of Maps Enables a Cytogenomics Analysis of the Complete Karyotype in Solea senegalensis.

The Pleuronectiformes order, which includes several commercially-important species, has undergone extensive chromosome evolution. One of these species is Solea senegalensis, a flatfish with 2n = 42 chromosomes. In this study, a cytogenomics approach and integration with previous maps was applied to characterize the karyotype of the species. Synteny analysis of S. senegalensis was carried out using two flatfish as a reference: Cynoglossus semilaevis and Scophthalmus maximus. Most S. senegalensis chromosomes (or chromosome arms for metacentrics and submetacentrics) showed a one-to-one macrosyntenic pattern with the other two species. In addition, we studied how repetitive sequences could have played a role in the evolution of S. senegalensis bi-armed (3, and 5–9) and acrocentric (11, 12 and 16) chromosomes, which showed the highest rearrangements compared with the reference species. A higher abundance of TEs (Transposable Elements) and other repeated elements was observed adjacent to telomeric regions on chromosomes 3, 7, 9 and 16. However, on chromosome 11, a greater abundance of DNA transposons was detected in interstitial BACs. This chromosome is syntenic with several chromosomes of the other two flatfish species, suggesting rearrangements during its evolution. A similar situation was also found on chromosome 16 (for microsatellites and low complexity sequences), but not for TEs (retroelements and DNA transposons). These differences in the distribution and abundance of repetitive elements in chromosomes that have undergone remodeling processes during the course of evolution also suggest a possible role for simple repeat sequences in rearranged regions.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly

Vanilla planifolia, the species cultivated to produce one of the world’s most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

Towards Predicting the Onset of Elastic Turbulence in Complex Geometries

Flow of complex fluids in porous structures is pertinent in many biological and industrial processes. For these applications, elastic turbulence, a viscoelastic instability occurring at low Re—arising from a non-trivial coupling of fluid rheology and flow geometry—is a common and relevant effect because of significant over-proportional increase in pressure drop and spatio-temporal distortion of the flow field. Therefore, significant efforts have been made to predict the onset of elastic turbulence in flow geometries with constrictions. The onset of flow perturbations to fluid streamlines is not adequately captured by Deborah and Weissenberg numbers. The introduction of more complex dimensionless numbers such as the M-criterion, which was meant as a simple and pragmatic method to predict the onset of elastic instabilities as an order-of-magnitude estimate, has been successful for simpler geometries. However, for more complex geometries which are encountered in many relevant applications, sometimes discrepancies between experimental observation and M-criteria prediction have been encountered. So far these discrepancies have been mainly attributed to the emergence from disorder. In this experimental study, we employ a single channel with multiple constrictions at varying distance and aspect ratios. We show that adjacent constrictions can interact via non-laminar flow field instabilities caused by a combination of individual geometry and viscoelastic rheology depending (besides other factors) explicitly on the distance between adjacent constrictions. This provides intuitive insight on a more conceptual level why the M-criteria predictions are not more precise. Our findings suggest that coupling of rheological effects and fluid geometry is more complex and implicit and controlled by more length scales than are currently employed. For translating bulk fluid, rheology determined by classical rheometry into the effective behaviour in complex porous geometries requires consideration of more than only one repeat element. Our findings open the path towards more accurate prediction of the onset of elastic turbulence, which many applications will benefit.Article HighlightsWe demonstrate that adjacent constrictions “interact” via the non-laminar flow fields caused by individual constrictions, implying that the coupling of rheological effects and fluid geometry is more complex and implicit.The concept of characterizing fluid rheology independent of flow geometry and later coupling back to the geometry of interest via dimensionless numbers may fall short of relevant length scales, such as the separation of constrictions which control the overlap of flow fields.By providing direct experimental evidence illustrating the cause of the shortcoming of the status-quo, the expected impact of this work is to challenge and augment existing concepts that will ultimately lead to the correct prediction of the onset of elastic turbulence.

From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.

Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.

Circle-Seq reveals genomic and disease-specific hallmarks in urinary cell-free extrachromosomal circular DNAs.

BackgroundExtrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine.MethodsHere, we report the discovery and analysis of urinary cell‐free eccDNAs (ucf‐eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle‐Seq.ResultsMillions of unique ucf‐eccDNAs were identified and comprehensively characterised. The ucf‐eccDNAs are GC‐rich. Most ucf‐eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf‐eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein‐coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf‐eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf‐eccDNA.ConclusionsThis work discovers and provides the first deep characterisation of ucf‐eccDNAs and suggests ucf‐eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Medicago truncatula (model legume), Medicago sativa (alfalfa), Medicago polymorpha (bur clover), and Medicago ruthenica

Alfalfa (Medicago sativa) is one of the most widely grown forage crops, providing a major protein source for livestock. In the tetraploid alfalfa genome, the four allelic chromosomes have a low level of sequence divergence, with similar sizes, numbers of genes, and repeat element contents, proving that autotetraploid alfalfa is a stable, random-pairing species.

Comparative Plastome Analysis of Three Amaryllidaceae Subfamilies: Insights into Variation of Genome Characteristics, Phylogeny, and Adaptive Evolution.

In the latest APG IV classification system, Amaryllidaceae is placed under the order of Asparagus and includes three subfamilies: Agapanthoideae, Allioideae, and Amaryllidoideae, which include many economically important crops. With the development of molecular phylogeny, research on the phylogenetic relationship of Amaryllidaceae has become more convenient. However, the current comparative analysis of Amaryllidaceae at the whole chloroplast genome level is still lacking. In this study, we sequenced 18 Allioideae plastomes and combined them with publicly available data (a total of 41 plastomes), including 21 Allioideae species, 1 Agapanthoideae species, 14 Amaryllidoideae species, and 5 Asparagaceae species. Comparative analyses were performed including basic characteristics of genome structure, codon usage, repeat elements, IR boundary, and genome divergence. Phylogenetic relationships were detected using single-copy genes (SCGs) and ribosomal internal transcribed spacer sequences (ITS), and the branch-site model was also employed to conduct the positive selection analysis. The results indicated that all Amaryllidaceae species showed a highly conserved typical tetrad structure. The GC content and five codon usage indexes in Allioideae species were lower than those in the other two subfamilies. Comparison analysis of Bayesian and ML phylogeny based on SCGs strongly supports the monophyly of three subfamilies and the sisterhood among them. Besides, positively selected genes (PSGs) were detected in each of the three subfamilies. Almost all genes with significant posterior probabilities for codon sites were associated with self-replication and photosynthesis. Our study investigated the three subfamilies of Amaryllidaceae at the whole chloroplast genome level and suggested the key role of selective pressure in the adaptation and evolution of Amaryllidaceae.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

DUX4 is a multifunctional factor priming human embryonic genome activation

SummaryDouble homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Chromosomal-Level Genome Assembly of the Springtail Tomocerus qinae (Collembola: Tomoceridae).

The family Tomoceridae is among the earliest derived collembolan lineages, thus is of key importance in understanding the evolution of Collembola. Here, we assembled a chromosome-level genome of one tomocerid species Tomocerus qinae by combining Nanopore long reads and Hi-C data. The final genome size was 334.44 Mb with the scaffold/contig N50 length of 71.85/13.94 Mb. BUSCO assessment indicated that 96.80% of complete arthropod universal single-copy orthologs (n = 1,013) were present in the assembly. The repeat elements accounted for 26.11% (87.26 Mb) and 494 noncoding RNAs were identified in the genome. A total of 20,451 protein-coding genes were predicted, which captured 96.0% (973) BUSCO genes. Gene family evolution analyses identified 4,825 expanded gene families of T. qinae, among them, 47 experienced significant expansions, and these significantly expanded gene families mainly involved in proliferation and growth. This study provides an important genomic resource for future evolution and comparative genomics analyses of Collembola.

Repeat Age Decomposition Informs an Ancient Set of Repeats Associated With Coleoid Cephalopod Divergence.

In comparison with other molluscs and bilaterians, the genomes of coleoid cephalopods (squid, cuttlefish, and octopus) sequenced so far show remarkably different genomic organization that presumably marked the early evolution of this taxon. The main driver behind this genomic rearrangement remains unclear. About half of the genome content in coleoids is known to consist of repeat elements; since selfish DNA is one of the powerful drivers of genome evolution, its pervasiveness could be intertwined with the emergence of cephalopod-specific genomic signatures and could have played an important role in the reorganization of the cephalopod genome architecture. However, due to abundant species-specific repeat expansions, it has not been possible so far to identify the ancient shared set of repeats associated with coleoid divergence. By means of an extensive repeat element re-evaluation and annotation combined with network sequence divergence approaches, we are able to identify and characterize the ancient repeat complement shared by at least four coleoid cephalopod species. Surprisingly, instead of the most abundant elements present in extant genomes, lower-copy-number DNA and retroelements were most associated with ancient coleoid radiation. Furthermore, evolutionary analysis of some of the most abundant families shared in Octopus bimaculoides and Euprymna scolopes disclosed within-family patterns of large species-specific expansions while also identifying a smaller shared expansion in the coleoid ancestor. Our study thus reveals the apomorphic nature of retroelement expansion in octopus and a conserved complement composed of several DNA element types and fewer LINE families.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks

BackgroundThe hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences.ResultsUsing a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves.ConclusionsThe diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

Long-read sequencing for identification of insertion sites in large transposon mutant libraries

Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long-Read Transposon Insertion-site Sequencing). LoRTIS enabled the unique localisation of transposon insertion sites within long repetitive genetic elements of E. coli, such as the transposase genes of insertion sequences and copies of the ~ 5 kb ribosomal RNA operon. We demonstrate that LoRTIS is reproducible, gives comparable results to short-read TIS methods for essential genes, and better resolution around repeat elements. The Oxford Nanopore sequencing device that we used is cost-effective, small and easily portable. Thus, LoRTIS is an efficient means of uniquely identifying transposon insertion sites within long repetitive genetic elements and can be easily transported to, and used in, laboratories that lack access to expensive DNA sequencing facilities.

Whole-genome sequence of the planarian Dugesia japonica combining Illumina and PacBio data.

Advances in stem cell biology have posed the challenges in revealing the mechanistic themes underlying whole tissues and organs formation during regeneration. The planarian Dugesia japonica is an ideal model organism for the study of regeneration and stem cell biology. However, the genome resources for this species are still limited. Here, we combined single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing, Illumina paired-end sequencing and 10× Genomics linked reads data to obtain the whole-genome sequence of the planarian D. japonica. The final assembled D. japonica genome is 1.13G with contig N50 of 248.44kb, and scaffold N50 of 652.52kb. Repeat elements account for 64.92% of the genome, and 12,031 protein coding genes were annotated, of which 10,114 genes had at least one functional annotation, representing 84.07% of the total genes. We present a highly contiguous genome assembly of D. japonica. The D. japonica genome assembly, together with gene annotation and transcriptome data provide a valuable resource to investigate molecular mechanism of planarian and stem cell research.

Genomic landscape, polymorphism and possible LINE-associated delivery of G-quadruplex motifs in the bovine genes.

G-Quadruplex structures are non-B DNA structures that occur in regions carrying short runs of guanines. They are implicated in several biological processes including transcription, translation, replication and telomere maintenance as well as in several pathological conditions like cancer and thus have gained the attention of the scientific community. The rise of the -omics era significantly affected the G-quadruplex research and the genome-wide characterization of G-Quadruplexes has been rendered a necessary first step towards applying genomics approaches for their study. While in human and several model organisms there is a considerable number of works studying genome-wide the DNA motifs with potential to form G-quadruplexes (G4-motifs), there is a total absence of any similar studies regarding livestock animals. The objectives of the present study were to provide a detailed characterization of the bovine genic G4-motifs' distribution and properties and to suggest a possible mechanism for the delivery of G4-motifs in the genes. Our data indicate that the distribution of G4-motifs within bovine genes and the annotation of said genes to Gene Ontology terms are similar to what is already shown for other organisms. By investigating their structural characteristics and polymorphism, it is obvious that the overall stability of the putative quadruplex structures is in line with the current notion in the G4 field. Similarly to human, the bovine G4-motifs are overrepresented in specific LINE repeat elements, the L1_BTs in the case of cattle. We highlight the potential role of these elements as vehicles for delivery of G4-motifs in the introns of the bovine genes. Lastly, it seems that a basis exists for connecting traits of agricultural importance to the genetic variation of G4-motifs, thus, the value of cattle as an interesting new model organism for G4-related genetic studies might be worth investigating.