Boom-Bust Turnovers of Megabase-Sized Centromeric DNA in Solanum Species: Rapid Evolution of DNA Sequences Associated with Centromeres

Centromeres are composed of long arrays of satellite repeats in most multicellular eukaryotes investigated to date. The satellite repeat–based centromeres are believed to have evolved from “neocentromeres” that originally contained only single- or low-copy sequences. However, the emergence and evolution of the satellite repeats in centromeres has been elusive. Potato (Solanum tuberosum) provides a model system for studying centromere evolution because each of its 12 centromeres contains distinct DNA sequences, allowing comparative analysis of homoeologous centromeres from related species. We conducted genome-wide analysis of the centromeric sequences in Solanum verrucosum, a wild species closely related to potato. Unambiguous homoeologous centromeric sequences were detected in only a single centromere (Cen9) between the two species. Four centromeres (Cen2, Cen4, Cen7, and Cen10) in S. verrucosum contained distinct satellite repeats that were amplified from retrotransposon-related sequences. Strikingly, the same four centromeres in potato contain either different satellite repeats (Cen2 and Cen7) or exclusively single- and low-copy sequences (Cen4 and Cen10). Our sequence comparison of five homoeologous centromeres in two Solanum species reveals rapid divergence of centromeric sequences among closely related species. We propose that centromeric satellite repeats undergo boom-bust cycles before a favorable repeat is fixed in the population.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka     

Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation). NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion. To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome. In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22. We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene. However, the GAP-related domain itself is not represented in this locus. In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21. These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus. Received: 18 December 1995 / Revised: 5 February 1996     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

Analysis of DNA restriction fragments greater than 5.7 Mb in size from the centromeric region of human chromosomes

Pulsed electrophoresis was used to study the organization of the human centromeric region. Genomic DNA was digested with rare-cutting enzymes. DNA fragments from 0.2 to greater than 5.7 Mb were separated by electrophoresis and hybridized with alphoid and simple DNA repeats. Rare-cutting enzymes (Mlu I, Nar I, Not I, Nru I, Sal I, Sfi I, Sst II) demonstrated fewer restriction sites at centromeric regions than elsewhere in the genome. The enzyme Not I had the fewest restriction sites at centromeric regions. As much as 70% of these sequences from the centromeric region are present in Not I DNA fragments greater than 5.7 and estimated to be as large as 10 Mb in size. Other repetitive sequences such as short interspersed repeated segments (SINEs), long interspersed repeated segments (LINEs), ribosomal DNA, and minisatellite DNA that are not enriched at the centromeric region, are not enriched in Not I fragments of greater than 5.7 Mb in size.     

Intragenomic movement,sequence amplification and concerted evolution in satellite DNA in harvest mice,Reithrodontomys: Evidence from in situ hybridization

Three DNA probes isolated from three species ofReithrodontomys (R. montanus, R. megalotis, R. fulvescens) were used to examine within and among species variation in the chromosomal location of satellite DNA and constitutive heterochromatin. These probes hybridized to the centromeric regions on all chromosomes in six species of the subgenusReithrodontomys. Additionally, nearly all extra-centromeric C-band positive regions (with the exception of some heterochromatic material on the X and Y) hybridized to these probes. Within the subgenusReithrodontomys both the chromosomal distribution and organization of satellite DNA has changed throughout evolution. The evolutionary transition has been from a totally centromeric position inR. fulvescens to centromeric and non-centromeric regions in other species that have undergone extensive chromosomal rearrangements from the primitive karyotype for peromyscine rodents. In addition, the monomer repeat of the satellite sequence differs betweenR. fulvescens (monomer defined by PstI) and the remaining species in the subgenusReithrodontomys (monomer defined by EcoRI). These results suggest at least two amplification events for this satellite DNA sequence. Models and mechanisms concerned with the homogenization and spread of satellite sequences in complex genomes are evaluated in light of theReithrodontomys data. From a phlyogenetic standpoint, the satellite sequences composing heterochromatic regions were restricted to the subgenusReithrodontomys, which supports morphological differences used to recognize two subgenera,Reithrodontomys andAporodon. Probes failed to hybridize to any part of the karyotype ofR. mexicanus (subgenusAporodon) or to seven species from other closely related genera (Baiomys, Neotoma, Nyctomys, Ochrotomys, Onychomys, Peromyscus, Xenomys), some of which are considered as potential sister taxa forReithrodontomys.     

The formation and evolution of centromeric satellite repeats in Saccharum species

Centromeres in eukaryotes are composed of tandem DNAs and retrotransposons. However, centromeric repeats exhibit considerable diversity, even among closely related species, and their origin and evolution are largely unknown. We conducted a genome-wide characterization of the centromeric sequences in sugarcane (Saccharum officinarum). Four centromeric tandem repeat sequences, So1, So103, So137 and So119, were isolated. So1 has a monomeric length of 137 bp, typical of a centromeric satellite, and has evolved four variants. However, these So1 variants had distinct centromere distributions and some were unique to an individual centromere. The distributions of the So1 variants were unexpectedly consistent among the Saccharum species that had different basic chromosome numbers or ploidy levels, thus suggesting evolutionary stability for approximately 7 million years in sugarcane. So103, So137 and So119 had unusually longer monomeric lengths that ranged from 327 to 1371 bp and lacked translational phasing on the CENH3 nucleosomes. Moreover, So103, So137 and So119 seemed to be highly similar to retrotransposons, which suggests that they originated from these mobile elements. Notably, all three repeats were flanked by direct repeats, and formed extrachromosomal circular DNAs (eccDNAs). The presence of circular molecules for these retrotransposon-derived centromeric satellites suggests an eccDNA-mediated centromeric satellite formation pathway in sugarcane.     

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.     

Physical location of a HSP70 gene homologue on the centromere of chromosome 1B of wheat ( Triticum aestivum L.)

Cereal centromeres consist of a complex organization of repetitive DNA sequences. Several repetitive DNA sequences are common amongst members of the Triticeae family, and others are unique to particular species. The organization of these repetitive elements and the abundance of other types of DNA sequences in cereal centromeres are largely unknown. In this study, we have used wheat-rye translocation lines to physically map 1BL.1RS centromeric breakpoints and molecular probes to obtain further information on the nature of other types of centromeric DNA sequences. Our results, using the rye-specific centromeric sequence, pAWRC.1, indicate that 1BL.1RS contains a small portion of the centromere from 1R of rye. Further studies used molecular markers to identify centromeric segments on wheat group-1 chromosomes. Selected RFLP markers, clustered around the centromere of wheat homoeologous group-1S chromosomes, were chosen as probes during Southern hybridization. One marker, PSR161, identified a small 1BS segment in all 1BL.1RS lines. This segment maps proximal to pAWRC.1 in 1BL.1RS and on the centromere of 1B. Sequence analysis of PSR161 showed high homology to HSP70 genes and Northern hybridization showed that this gene is constitutively expressed in leaf tissue and induced by heat shock and light stimuli. The significance of this work with respect to centromere organization and the possible significance of this HSP70 gene homologue are discussed. Received: 12 March 2001 / Accepted: 14 June 2001     

Genus-specific localization of the TaiI family of tandem-repetitive sequences in either the centromeric or subtelomeric regions in Triticeae species (Poaceae) and its evolution in wheat.

The TaiI family sequences are classified as tandem repetitive DNA sequences present in the genome of tribe Triticeae, and are localized in the centromeric regions of common wheat, but in the subtelomeric heterochromatic regions of Leymus racemosus and related species. In this study, we investigated the chromosomal distribution of TaiI family sequences in other Triticeae species. The results demonstrated a centromeric localization in genera Triticum and Aegilops and subtelomeric localization in other genera, thus showing a genus-dependent localization of TaiI family sequences in one or the other region. The copy numbers of TaiI family sequences in species in the same genus varied greatly, whether in the centromeric or subtelomeric regions (depending on genus). We also examined the evolution of TaiI family sequences during polyploidization of hexaploid common wheat. A comparison of chromosomal locations of the major TaiI family signals in common wheat and in its ancestral species suggested that the centromeric TaiI family sequences in common wheat were inherited from its ancestors with little modification, whereas a mixed origin for the B genome of common wheat was indicated.     

Structure and dynamics of retrotransposons at wheat centromeres and pericentromeres

Little is known of the dynamics of centromeric DNA in polyploid plants. We report the sequences of two centromere-associated bacterial artificial chromosome clones from a Triticum boeoticum library. Both autonomous and non-autonomous wheat centromeric retrotransposons (CRWs) were identified, both being closely associated with the centromeres of wheat. Fiber-fluorescence in situ hybridization and chromatin immunoprecipitation analysis showed that wheat centromeric retrotransposons (CRWs) represent a dominant component of the wheat centromere and are associated with centromere function. CRW copy number showed variation among different genomes: the D genome chromosomes contained fewer copies than either the A or B genome chromosomes. The frequency of lengthy continuous CRW arrays was higher than that in either rice or maize. The dynamics of CRWs and other retrotransposons at centromeric and pericentromeric regions during diploid speciation and polyploidization of wheat and its related species are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zhao Liu and Wei Yue made an equal contribution to this work.     

Characterization and chromosomal location of two repeated DNAs in three Gerbillus species

Two tandemly repeated DNA sequences of Gerbillus nigeriae (Rodentia) (GN1 and GN2) were isolated and characterized. Both share a 36 bp repeated unit, which includes a 20 bp motif also found in primate alphoid and other repeated DNAs. The localization of GN1 and GN2 sequences on metaphase chromosomes of three Gerbillus species, G. nigeriae, G. aureus and G. nanus, was studied by fluorescence in situ hybridization (FISH). In the G. nigeriae and G. aureus karyotypes, which were shown to possess large amounts of heterochromatin and to have undergone multiple rearrangements during evolution, both GN1 and GN2 sequences were observed at various chromosomal sites: centromeric, telomeric and intercalary. In contrast, the karyotypically stable G. nanus, which does not possess large amounts of heterochromatin and seems to be a more ancestral species, possesses only GN1 sequences, localized in the juxtacentromeric regions.     

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in α-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster. Received: 13 April 1996; in revised form: 25 June 1996 / Accepted: 6 July 1996     

Heterochromatic DNA in Triturus (Amphibia,Urodela)

The MspI family of highly repeated sequences is a centromeric satellite DNA representing about 1% of the genome of the Italian smooth newt, Triturus vulgaris meridionalis. We have studied the structure, genomic organization, chromosomal localization and conservation across species of this family. MspI sequences are around 197 bp long, as shown by sequencing of three cloned units. The family is organized in large clusters of tandemly arrayed units, present at almost all the centromeres of T.v. meridionalis, and is well conserved in the T.v. vulgaris subspecies. Conserved MspI sequences are also present in the related species T. helveticus, where they appear to be clustered at the centromeres of only a few chromosomes. MspI sequences are not found in other Triturus species analysed. The correlation of these sequences with the overall distribution pattern of heterochromatin and the extent of their conservation within the genus Triturus, are discussed.     

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D‐genome) of the allopolyploid (AD‐genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin‐immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon‐related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A‐genome and related diploid species (B‐, F‐ and G‐genomes), indicating that they colonized the centromeres of D‐genome lineage after the divergence of the A‐ and D‐ ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A‐ and D‐subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D‐ and AD‐genome species, yet localized to just the NORs in A‐, B‐, F‐, and G‐genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.     

Strong nucleosomes of mouse genome including recovered centromeric sequences

Recently discovered strong nucleosomes (SNs) characterized by visibly periodical DNA sequences have been found to concentrate in centromeres of Arabidopsis thaliana and in transient meiotic centromeres of Caenorhabditis elegans. To find out whether such affiliation of SNs to centromeres is a more general phenomenon, we studied SNs of the Mus musculus. The publicly available genome sequences of mouse, as well as of practically all other eukaryotes do not include the centromere regions which are difficult to assemble because of a large amount of repeat sequences in the centromeres and pericentromeric regions. We recovered those missing sequences using the data from MNase-seq experiments in mouse embryonic stem cells, where the sequence of DNA inside nucleosomes, including missing regions, was determined by 100-bp paired-end sequencing. Those nucleosome sequences, which are not matching to the published genome sequence, would largely belong to the centromeres. By evaluating SN densities in centromeres and in non-centromeric regions, we conclude that mouse SNs concentrate in the centromeres of telocentric mouse chromosomes, with ~3.9 times excess compared to their density in the rest of the genome. The remaining non-centromeric SNs are harbored mainly by introns and intergenic regions, by retro-transposons, in particular. The centromeric involvement of the SNs opens new horizons for the chromosome and centromere structure studies.