Andrea Koblížková

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

The authors used a genome-wide approach to identify the DNA sequences associated with the centromeres of potato chromosomes. Five potato centromeres are composed of single- or low-copy sequences, including active genes; six centromeres contain almost exclusively satellite repeats. The results have implications for evolution of repeat-based centromeres from repeatless centromeres. 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.

Plant centromeric retrotransposons: a structural and cytogenetic perspective

BackgroundThe centromeric and pericentromeric regions of plant chromosomes are colonized by Ty3/gypsy retrotransposons, which, on the basis of their reverse transcriptase sequences, form the chromovirus CRM clade. Despite their potential importance for centromere evolution and function, they have remained poorly characterized. In this work, we aimed to carry out a comprehensive survey of CRM clade elements with an emphasis on their diversity, structure, chromosomal distribution and transcriptional activity.ResultsWe have surveyed a set of 190 CRM elements belonging to 81 different retrotransposon families, derived from 33 host species and falling into 12 plant families. The sequences at the C-terminus of their integrases were unexpectedly heterogeneous, despite the understanding that they are responsible for targeting to the centromere. This variation allowed the division of the CRM clade into the three groups A, B and C, and the members of each differed considerably with respect to their chromosomal distribution. The differences in chromosomal distribution coincided with variation in the integrase C-terminus sequences possessing a putative targeting domain (PTD). A majority of the group A elements possess the CR motif and are concentrated in the centromeric region, while members of group C have the type II chromodomain and are dispersed throughout the genome. Although representatives of the group B lack a PTD of any type, they appeared to be localized preferentially in the centromeres of tested species. All tested elements were found to be transcriptionally active.ConclusionsComprehensive analysis of the CRM clade elements showed that genuinely centromeric retrotransposons represent only a fraction of the CRM clade (group A). These centromeric retrotransposons represent an active component of centromeres of a wide range of angiosperm species, implying that they play an important role in plant centromere evolution. In addition, their transcriptional activity is consistent with the notion that the transcription of centromeric retrotransposons has a role in normal centromere function.

Development and Characterization of Microsatellite Markers from Chromosome 1-Specific DNA Libraries of Vicia Faba

An integrated approach has been developed for targeted retrieval of microsatellite markers from selected regions of the field bean (Vicia faba L.) genome. The procedure is based on a combination of advanced physical and genetic mapping techniques and includes the following steps: 1) flow-sorting of metaphase chromosomes, 2) construction of microsatellite-enriched chromosome-specific DNA libraries, 3) isolation of polymorphic microsatellite sequences from the libraries, 4) testing chromosome specificity of the microsatellites using polymerase chain reaction with purified fractions of individual chromosome types, and 5) integration of chromosome-specific markers into a genetic map. Several strategies for isolation of microsatellite clones were tested, including direct screening of non-enriched libraries with single or mixed probes and screening of the libraries after one or two rounds of enrichment. Finally, the usefulness of this approach was demonstrated by the retrieval of novel markers from a selected portion of the largest field been chromosome (No. 1).

Next Generation Sequencing-Based Analysis of Repetitive DNA in the Model Dioceous Plant Silene latifolia

Background Silene latifolia is a dioceous plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome. Methodology/Principal Findings We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data. Conclusions/Significance This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3–5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel “meta-polycentric” functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

Survey of extrachromosomal circular DNA derived from plant satellite repeats

BackgroundSatellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.ResultsWe performed a survey of eccDNA corresponding to nine different families and three subfamilies of satellite repeats in ten species from various genera of higher plants (Arabidopsis, Oryza, Pisum, Secale, Triticum and Vicia). The repeats selected for this study differed in their monomer length, abundance, and chromosomal localization in individual species. Using two-dimensional agarose gel electrophoresis followed by Southern blotting, eccDNA molecules corresponding to all examined satellites were detected. EccDNA occurred in the form of nicked circles ranging from hundreds to over eight thousand nucleotides in size. Within this range the circular molecules occurred preferentially in discrete size intervals corresponding to multiples of monomer or higher-order repeat lengths.ConclusionThis work demonstrated that satellite repeat-derived eccDNA is common in plant genomes and thus it can be seriously considered as a potential intermediate in processes driving satellite repeat evolution. The observed size distribution of circular molecules suggests that they are most likely generated by molecular mechanisms based on homologous recombination requiring long stretches of sequence similarity.

Diverse retrotransposon families and an AT-rich satellite DNA revealed in giant genomes of Fritillaria lilies.

BACKGROUND AND AIMS The genus Fritillaria (Liliaceae) comprises species with extremely large genomes (1C = 30 000-127 000 Mb) and a bicontinental distribution. Most North American species (subgenus Liliorhiza) differ from Eurasian Fritillaria species by their distinct phylogenetic position and increased amounts of heterochromatin. This study examined the contribution of major repetitive elements to the genome obesity found in Fritillaria and identified repeats contributing to the heterochromatin arrays in Liliorhiza species. METHODS Two Fritillaria species of similar genome size were selected for detailed analysis, one from each phylogeographical clade: F. affinis (1C = 45·6 pg, North America) and F. imperialis (1C = 43·0 pg, Eurasia). Fosmid libraries were constructed from their genomic DNAs and used for identification, sequence characterization, quantification and chromosome localization of clones containing highly repeated sequences. KEY RESULTS AND CONCLUSIONS Repeats corresponding to 6·7 and 4·7 % of the F. affinis and F. imperialis genome, respectively, were identified. Chromoviruses and the Tat lineage of Ty3/gypsy group long terminal repeat retrotransposons were identified as the predominant components of the highly repeated fractions in the F. affinis and F. imperialis genomes, respectively. In addition, a heterogeneous, extremely AT-rich satellite repeat was isolated from F. affinis. The FriSAT1 repeat localized in heterochromatic bands makes up approx. 26 % of the F. affinis genome and substantial genomic fractions in several other Liliorhiza species. However, no evidence of a relationship between heterochromatin content and genome size variation was observed. Also, this study was unable to reveal any predominant repeats which tracked the increasing/decreasing trends of genome size evolution in Fritillaria. Instead, the giant Fritillaria genomes seem to be composed of many diversified families of transposable elements. We hypothesize that the genome obesity may be partly determined by the failure of removal mechanisms to counterbalance effectively the retrotransposon amplification.

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

This work compares the sequences of five homoeologous centromeres in two closely related species, revealing rapid divergence of centromeric sequences. The results support the idea that centromeric satellite repeats undergo boom-bust cycles before a favorable repeat is fixed in the population. 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.

Genome-wide analysis of repeat diversity across the family Musaceae.

Background The banana family (Musaceae) includes genetically a diverse group of species and their diploid and polyploid hybrids that are widely cultivated in the tropics. In spite of their socio-economic importance, the knowledge of Musaceae genomes is basically limited to draft genome assemblies of two species, Musa acuminata and M. balbisiana. Here we aimed to complement this information by analyzing repetitive genome fractions of six species selected to represent various phylogenetic groups within the family. Results Low-pass sequencing of M. acuminata, M. ornata, M. textilis, M. beccarii, M. balbisiana, and Ensete gilletii genomes was performed using a 454/Roche platform. Sequence reads were subjected to analysis of their overall intra- and inter-specific similarities and, all major repeat families were quantified using graph-based clustering. Maximus/SIRE and Angela lineages of Ty1/copia long terminal repeat (LTR) retrotransposons and the chromovirus lineage of Ty3/gypsy elements were found to make up most of highly repetitive DNA in all species (14–34.5% of the genome). However, there were quantitative differences and sequence variations detected for classified repeat families as well as for the bulk of total repetitive DNA. These differences were most pronounced between species from different taxonomic sections of the Musaceae family, whereas pairs of closely related species (M. acuminata/M. ornata and M. beccarii/M. textilis) shared similar populations of repetitive elements. Conclusions This study provided the first insight into the composition and sequence variation of repetitive parts of Musaceae genomes. It allowed identification of repetitive sequences specific for a single species or a group of species that can be utilized as molecular markers in breeding programs and generated computational resources that will be instrumental in repeat masking and annotation in future genome assembly projects.

Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution.