Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia)

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30–42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG) _n sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.     

Conservation of Human Gamma-X Centromeric Satellite DNA Among Primates with an Autosomal Localization in Certain Old World monkeys

Gamma-X satellite DNA is a 220-bp tandemly arranged repetitive DNA with specificity for the centromeric region of the human X chromosome. The conservation of this human X centromeric satellite DNA sequence in primate species was evaluated by comparative fluorescence in-situ hybridization to metaphase chromosome preparations of the great apes and three Old World monkeys. Homologous gamma-X DNA were detected at centromeric locations in all six primate species. For the great apes, gamma-X was exclusively localized to the centromeric regions of the X chromosomes. Among the Old World monkeys studied, only the golden monkey exhibited localization to the X chromosome. In the black-and-white colobus and the pig-tailed macaque, human gamma-X sequences were localized to the pericentromeric regions of autosomes 1 and 4, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes

A distinctive feature of the avian genome is the large heterogeneity in the size of chromosomes, which are usually classified into a small number of macrochromosomes and numerous microchromosomes. These chromosome classes show characteristic differences in a number of interrelated features that could potentially affect the rate of sequence evolution, such as GC content, gene density, and recombination rate. We studied the effects of these factors by analyzing patterns of nucleotide substitution in two sets of chicken-turkey sequence alignments. First, in a set of 67 orthologous introns, divergence was significantly higher in microchromosomes (chromosomes 11-38; 11.7% divergence) than in both macrochromosomes (chromosomes 1-5; 9.9% divergence; P = 0.016) and intermediate-sized chromosomes (chromosomes 6-10; 9.5% divergence; P = 0.026). At least part of this difference was due to the higher incidence of CpG sites on microchromosomes. Second, using 155 orthologous coding sequences we noted a similar pattern, in which synonymous substitution rates on microchromosomes (13.1%) were significantly higher than were rates on macrochromosomes (10.3%; P = 0.024). Broadly assuming neutrality of introns and synonymous sites, or constraints on such sequences do not differ between chromosomal classes, these observations imply that microchromosomal genes are exposed to more germ line mutations than those on other chromosomes. We also find that dN/dS ratios for genes located on microchromosomes (average, 0.094) are significantly lower than those of macrochromosomes (average, 0.185; P = 0.025), suggesting that the proteins of genes on microchromosomes are under greater evolutionary constraint.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

Comparative Painting Reveals Strong Chromosome Homology Over 80 Million Years of Bird Evolution

Chickens and the great flightless emu belong to two distantly related orders of birds in the carinate and ratite subclasses that diverged at least 80 million years ago. In the first ZOO-FISH study between bird species, we hybridized single chromosome paints from the chicken (Gallus domesticus) onto the emu chromosomes. We found that the nine macrochromosomes show remarkable homology between the two species, indicating strong conservation of karyotype through evolution. One chicken macrochromosome (4) was represented by a macro- and a microchromosome in the emu, suggesting that microchromosomes and macrochromosomes are interconvertible. The chicken Z chromosome paint hybridized to the emu Z and most of the W, confirming that ratite sex chromosomes are largely homologous; the centromeric region of the W which hybridized weakly may represent the location of the sex determining gene(s).     

A Novel Repetitive Sequence of Sugar Cane, SCEN Family, Locating on Centromeric Regions

Tandem repetitive sequences consisting of 140-bp repetitive units were cloned from sugar cane genomic DNA and designated the SCEN family. In situ hybridization revealed that they were located on the centromeric region of almost all of the chromosomes of sugar cane. The 140-bp sequence included three CENP-B box-like sequences. Phylogenetic analysis of the members of the SCEN family revealed that the sequences had 75% homology with each other, on average, and that the sequences could not be further classified into smaller subfamilies. The copy number of the sequence was estimated to be 2.6 × 10⁵ per haploid sugar cane genome and, therefore, 4.6 × 10³ or 630 kb per chromosome on average.     

Partially Inverted Tandem Repeat Isolated from Pericentric Region of Chicken Chromosome 8

The majority of chicken repetitive sequence is nuclear-membrane-associated sequence (CNM), which resides in a large number of microchromosomes (chromosomes 11–39) and is absent from macrochromosomes 1–5, ZW, and some of the intermediate chromosomes 6–10. Two repetitive families, EcoRI/XhoI, are confined to the female-specific W chromosome. The core repeat units of the three families are 21 bp, containing (A)_3–5 and (T)_3–5 clusters separated by 5–7-bp sequences. In this article, we describe the isolation and initial characterization of a novel repeat family that is related to CNM/EcoRI/XhoI families. The novel family, designated as PIR, consists of multiple types of partially inverted repeat units of about 1.2, 1.4 and 1.6 kb. The PIR sequence is restricted to chicken chromosome 8, and accounts for about 3.8 mb, or 2500 copies of the 1.4-kb units, of the chicken genome. The evolution of PIR and related sequences is discussed.     

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (<Emphasis Type="Italic">Apodemus peninsulae</Emphasis>, Muridae,Rodentia)

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.     

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.     

RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: Comparison with vaccine virus and field isolates from England,Scotland and the Republic of Ireland

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.     

Clinical Application of PCR-Restriction Enzyme Pattern Analysis for Rapid Identification of Aerobic Actinomycete Isolates

The accuracy and practicality of PCR-restriction enzyme pattern analysis (PRA) for routine identification of aerobic actinomycete clinical isolates were evaluated for 299 cultures submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler. PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by traditional methods, including growth characteristics, susceptibility patterns, biochemical testing, and high-performance liquid chromatography analysis. Microbiological examination of six cultures ruled out aerobic actinomycetes, and they were omitted from the study. Amplicons were analyzed with BstEII, HaeIII, MspI, HinfI, and BsaHI. When necessary, AciI, HhaI, and NarI were also used. From March 1995 through May 1997 (27 months), 274 of the remaining 293 (93.5%) isolates were accurately identified by PRA. Major diagnostic groups included 170 mycobacteria, 93 nocardiae, and 30 other aerobic actinomycetes. Mixed cultures were readily recognized by PRA, including a wound culture that contained two Nocardia taxa that were indistinguishable morphologically. Mycobacterium mucogenicum was identified in three cultures heavily contaminated with gram-positive cocci. The 19 isolates that produced PRA patterns that did not match those in the current PRA database were differentiated into 8 Mycobacterium species and 11 other aerobic actinomycetes by the presence or absence of BstEII recognition sites. Identification of 15 of these 19 isolates was also equivocal by traditional methods. PRA results were reportable within 2 to 5 working days and were as accurate as and faster and less expensive to obtain than those of traditional methods.     

Conservation of a 31-bp bovine subrepeat in centromeric satellite DNA monomers ofCervus elaphus and other cervid species

A centromeric satellite DNA clone was isolated from the genome of the European red deer (Cervus elaphus hippelaphus) and designated Ce-Pst1. This clone was localized to the centromeric region of all red deer chromosomes with the exception of a single pair of metacentric autosomes and the Y chromosome. DNA sequence analysis of the 806-bp Ce-Pst1 clone showed 73.0–78.9% sequence homology to four previously isolated cervid centromeric satellite DNA clones, suggesting that the Ce-Pst1 clone is yet another member of the major cervid centromeric satellite DNA family. Using a DNA sequence comparison system, internal 31-bp tandem subrepeats were found in the Ce-Pst1 clone as well as in the other previously reported cervid centromeric satellite DNA monomer sequences. A 31-bp consensus sequence was constructed for each cervid monomer clone and shown to be highly homologous to the 31-bp subrepeat consensus sequence found in bovine 1.715 centromeric satellite DNA. The identification of internal subrepeats in the satellite monomers studied could suggest that amplification of an ancestral 31-bp DNA sequence may have contributed to the genesis of major cervid centromeric satellite DNA. The homology between the 31-bp subrepeats found in cervid and bovid centromeric satellite DNAs substantiates the theory that amplification of this 31-bp DNA sequence may have occurred before the evolutionary separation of these two families 20–25 million years ago.accepted for publication by H. C. Macgregor     

Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution

In-situ hybridization with a rDNA probe has demonstrated the presence of non-transcribed ribosomal genes in the B chromosomes of the black rat Rattus rattus. To test whether methylation of ribosomal DNA present in the B chromosomes could account for their inactivation, we performed in-situ digestions and Southern analyses of DNA digested with the isoschizomers MspI and HpaII. Our results suggest that the accessory chromosomes of this species have originated from one of the smaller NOR-carrying chromosome pairs. In the course of evolution, repetitive sequences invaded this supernumerary element and its ribosomal DNA content was dispersed throughout the chromosome and inactivated by heterochromatinization and methylation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Centromeric heterochromatin in the cattle rob(1;29) translocation: α-satellite I sequences, in-situ MspI digestion patterns, chromomycin staining and C-bands

The centromeric regions and -satellite I sequence were studied on chromosomes 1, 29 and the rob(1;29) translocation in a Portuguese breed of cattle, Barrosã, carrying the translocation. Rob(1;29) centromeric regions showed heterochromatic bands with propidium iodide but, unlike the acrocentric autosomes, no strong centromeric bands were revealed with chromomycin A3. An -satellite I sequence was not found at the centromeres of the X, Y and rob(1;29) chromosomes in the breed, although it was present at the centromeres of all acrocentric chromosomes including 1 and 29. Restriction enzyme banding with MspI revealed polymorphisms between different rob(1;29) chromosomes in both centromeric and intercalary regions. The data show that the centromeric region of the rob(1;29) chromosome has lost the -satellite I sequences, while retaining other heterochromatin, and suggest that this important and widespread translocation has occurred multiple times.     

Practical Approach for Detection and Identification of OXA-10-Derived Ceftazidime-Hydrolyzing Extended-Spectrum β-Lactamases

A practical approach to detect and identify ceftazidime-hydrolyzing extended-spectrum mutants of OXA-10 β-lactamase is presented. Large numbers of bacteria were screened by colony hybridization, a 720-bp part of bla_OXA was amplified by PCR from the hybridization-positive isolates, and the products were digested by PvuII and HaeIII.     

Mitochondrial DNA polymorphism in the Vietnamese population

The mitochondrial DNA variation was screened in a sample of 50 unrelated individuals of the Vietnamese population originating from Hanoi. A combination of long and standard PCR and restriction endonuclease digests with the enzymes HpaI, BamHI, HaeII, MspI, AvaII and HincII were used to reveal mtDNA variation. Twenty enzyme morphs were detected, three of which (HaeII-13^Viet, MspI-19^Viet and MspI-20^Viet) are new and are produced by a single mutational event in already known enzyme morphs. Ten already known and four new mitotypes [93^Viet (1-1-2-4-1), 94^Viet (2-1-13^Viet-1-1), 95^Viet (2-1-13^Viet-19^Viet-1) and 96^Viet (1-1-2–20^Viet-12)] were found in the Vietnamese population. The 9-bp deletion occurring in the COII/tRNA^Lys region of the mitochondrial genome was also analysed and 10 samples were found to have this deletion. The comparison of the Vietnamese with other East Asian populations showed a close genetic relationship of the population under investigation with other Orientals. However, the Vietnamese population can be differentiated by the significantly higher frequency of the enzyme morph HincII-5 and by seven new markers. These results strongly support the hypothesis of a dual ethnic origin of the Vietnamese population from the Chinese and Thai–Indonesian populations based on HLA markers and linguistic evidence.     

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.     

Cytogenetic comparison of heteromorphic and homomorphic sex chromosomes in <Emphasis Type="Italic">Coccinia</Emphasis> (Cucurbitaceae) points to sex chromosome turnover

Our understanding of the evolution of plant sex chromosomes is increasing rapidly due to high-throughput sequencing data and phylogenetic and molecular-cytogenetic approaches that make it possible to infer the evolutionary direction and steps leading from homomorphic to heteromorphic sex chromosomes. Here, we focus on four species of Coccinia, a genus of 25 dioecious species, including Coccinia grandis, the species with the largest known plant Y chromosome. Based on a phylogeny for the genus, we selected three species close to C. grandis to test the distribution of eight repetitive elements including two satellites, and several plastid and mitochondrial probes, that we had previously found to have distinct accumulation patterns in the C. grandis genome. Additionally, we determined C-values and performed immunostaining experiments with (peri-)centromere-specific antibodies on two species (for comparison with C. grandis). In spite of no microscopic chromosomal heteromorphism, single pairs of chromosomes in male cells of all three species accumulate some of the very same repeats that are enriched on the C. grandis Y chromosome, pointing to either old (previous) sex chromosomes or incipient (newly arising) ones, that is, to sex chromosome turnover. A 144-bp centromeric satellite repeat (CgCent) that characterizes all C. grandis chromosomes except the Y is highly abundant in all centromeric regions of the other species, indicating that the centromeric sequence of the Y chromosome diverged very recently.     

Identification and characterization of centromeric sequences in Xenopus laevis

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant Centromere Protein A (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences, but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

Accurate chromosome segregation during cell division requires the centromere, a nucleoprotein complex assembled on each chromosome that is essential for chromosome segregation. Centromeres provide the assembly site for the mitotic kinetochore that mediates microtubule attachment and error correction during mitosis (Foley and Kapoor 2013). Centromeres are defined epigenetically by the histone H3 variant, Centromere Protein A (Cenpa), the presence of which is both necessary and sufficient for centromere formation (Musacchio and Desai 2017). Unlike histone H3.1 nucleosomes, that are assembled as chromosomes replicate in S-phase, Cenpa nucleosomes are replenished after replication during the next G1 phase of the cell cycle. Cenpa nucleosomes in chromatin appear to epigenetically dictate the sites of new Cenpa incorporation, thereby providing a mechanism for self-maintenance (Zasadzińska and Foltz 2017).In humans, centromeres form on tandem repeats of an ∼171-bp DNA sequence termed ɑ-satellite. Each 171-bp monomer shares ∼60% sequence homology with other monomers. Tandem arrays of monomers are repeated in blocks of higher order repeats (HORs), resulting in long stretches of virtually identical repeat sequences (Willard and Waye 1987; Rudd et al. 2003; McNulty and Sullivan 2018). Investigation into the genetic features required to form stable human artificial chromosomes (HACs) identified repetitive ɑ-satellite DNA as sufficient for de novo centromere formation when introduced into human cells (Harrington et al. 1997; Ohzeki et al. 2015). These studies demonstrated that repetitive DNA promotes centromere formation in vertebrates.Perturbing centromere function in cells often leads to cell death; thus, cell-free systems using budding yeast and Xenopus laevis egg extracts have been invaluable for studying centromere and kinetochore assembly (Ng and Carbon 1987; Hyman et al. 1992; Sorger et al. 1994; Desai et al. 1997; Akiyoshi et al. 2010; Guse et al. 2011; Moree et al. 2011). Budding yeast centromeres are defined by a single 125-bp DNA sequence that is sufficient to recruit much of the centromere and kinetochore in cell extracts. As in humans, Xenopus laevis builds its centromeres on repetitive sequences (Edwards and Murray 2005), and thus Xenopus egg extract provides a unique system to study the functions of repetitive DNA in driving centromere formation. A 174-bp centromeric repeat has been previously identified in Xenopus laevis by chromatin immunoprecipitation of Cenpa followed by cloning and sequencing (Edwards and Murray 2005). This repeat sequence termed Frog centromere repeat 1 (Fcr1) forms large repetitive arrays and is AT rich, as are centromeric repeats from other vertebrates (Manuelidis 1978; McDermid et al. 1986; Melters et al. 2013; Sullivan et al. 2017). Fcr1 is detected on only 60%–70% of Xenopus laevis centromeres, suggesting that there must be other sequence elements that comprise Xenopus laevis centromeres. Xenopus laevis is an allotetraploid species: the genome is composed of two related subgenomes named the long (L) and short (S) based on the length of the homoeologous chromosomes (Session et al. 2016). Whether there is conservation of centromeric repeats within each subgenome or between homoeologous chromosomes remains unknown.In this study, we identified and characterized Cenpa-associated sequences in Xenopus laevis. We utilized a k-mer-based method that does not depend on an assembled reference genome and thus provides an unbiased approach to identify sequence motifs present at the centromere. Our results demonstrate the sequence diversity at active Xenopus laevis centromeres and enable future studies of the function of repetitive elements in centromere formation and function.