Retrotransposons consist of significant portions of many complex eukaryotic genomes and are often... more Retrotransposons consist of significant portions of many complex eukaryotic genomes and are often enriched in heterochromatin. The centromeric retrotransposon (CR) family in grass species is colonized in the centromeres and highly conserved among species that have been diverged for .50 MY. These unique characteristics have inspired scientists to speculate about the roles of CR elements in organization and function of
Continuously growing hairy-root cultures of pea (Pisum sativum L.) line L-84 have been induced by... more Continuously growing hairy-root cultures of pea (Pisum sativum L.) line L-84 have been induced by transformation of seedlings by Agrobacterium rhizogenes A4-24. The cultures were then used for cell cycle synchronisation in vitro and for preparation of chromosome suspensions. Various concentrations of hydroxyurea (0.5–2.0 mM) and timing of amiprophos-methyl (APM, 2–4 h) treatment have been tested to achieve the highest mitotic synchrony, and to accumulate the highest number of synchronised cells in metaphase, respectively. Optimal conditions (2 mM hydroxyurea for 18 h, 10 μM APM for 2 h applied 6 h after a release from hydroxyurea block) resulted in a high frequency of cells in metaphase (43%). The synchronised root tips were fixed in formaldehyde and chromosomes were released into a lysis buffer by mechanical homogenisation. The chromosomes in suspension showed a well preserved morphology, and could be used for flow cytometric analysis. While only two chromosomes (5 and 7) were discriminated in a standard karyotype, four chromosomes (3, 5, 6 and 7) could be clearly discriminated in the line L-84 which contains a stable reciprocal translocation between chromosomes 3 and 6. The establishment of hairy root cultures and the development of an efficient chromosome isolation procedure from a translocation line L-84 represent a basic prerequisite for subsequent flow-sorting of selected chromosomes of pea.
Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structu... more Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structure of their centromeric regions, which is clearly apparent during metaphase by the formation of extended primary constrictions which span up to a third of the length of the chromosome. In addition, these species express two different variants of the CenH3 protein which are co-localized in multiple domains along the poleward surface of the primary constrictions. Here, we show that the constrictions represent a distinct type of chromatin differing from the chromosome arms. In metaphase, histone phosphorylation patterns including H3S10ph, H3S28ph, and H3T3ph were observed along the entire constriction, in a way similar to holocentric chromosomes. On the other hand, distribution of phosphorylated H2AT120 was different from that previously reported from either, holocentric and monocentric chromosomes, occurring at chromatin surrounding but not overlapping CenH3 domains. Since some of these phosphorylations play a role in chromatid cohesion, it can be assumed that they facilitate correct chromosome segregation by ensuring that multiple separate CenH3 domains present on the same chromatid are oriented toward the same pole. The constrictions also displayed distinct patterns of histone methylation marks, being enriched in H3K9me2 and depleted in H3K4me3 and H3K27me2 compared to the chromosome arms. Super-resolution fluorescence microscopy revealed that although both CenH3 protein variants are present in all CenH3 domains detected on metaphase chromosomes, they are only partially co-localized while there are chromatin subdomains which are mostly made of only one CenH3 variant. Taken together, these data revealed specific features of extended primary constrictions of Lathyrus and Pisum and support the idea that they may represent an intermediate stage between monocentric and holocentric chromosomes.
The differential accumulation and elimination of repetitive DNA are key drivers of genome size va... more The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encou...
Proceedings of the National Academy of Sciences, 2015
Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinet... more Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.
The Plant journal : for cell and molecular biology, Jan 20, 2015
Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure... more Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra di...
Centromeres are essential structures for proper chromosome segregation during cell division. Howe... more Centromeres are essential structures for proper chromosome segregation during cell division. However, the relationship between centromere structure, sequence composition and function is still unclear. To study these questions, we adopted a model system of two closely related pea species. Garden pea (Pisum sativum) mitotic chromosomes harbor unique centromeres with multiple functional domains embedded within an extended primary constriction. Also, the sequence composition of the pea centromere differs from any other species studied so far. Pea centromeres are composed of thirteen families of satellite repeats differing in their monomer length. None of the families are present on all chromosomes and individual CenH3 domains on a single chromosome often contain different repeats. Using CenH3 antibody, we detected elongated centromeres also in some of the chromosomes of tawny pea (P. fulvum), a wild relative of the garden pea. To identify repetitive sequences in both genomes, we employe...
Centromeric regions of plant chromosomes are usually made of complex populations of repetitive el... more Centromeric regions of plant chromosomes are usually made of complex populations of repetitive elements which are difficult to investigate using conventional cloning and sequencing approaches. In addition, requirements for large-scale sequencing and the lack of suitable bioinformatics tools have long hampered centromere investigation in non-model species. In this lecture, we will present an experimental workflow that takes advantage of recently introduced next generation sequencing technologies and newly developed computational tools to overcome these limitations and allow investigation of centromere composition in a wide range of plant species. Combining whole genome and CenH3-ChIP sequencing with data analysis using RepeatExplorer pipeline led to the identification of all major centromeric repeats in cultivated pea (Pisum sativum) and its wild relative P. fulvum. Both species have extended centromeres which are composed of multiple CenH3 domains associated with more than ten diffe...
The centromere is a functional chromosome domain that is essential for faithful chromosome segreg... more The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division. It can be reliably identified by the presence of CenH3 histone. In most diploid plant genomes, CenH3 is encoded by a single copy gene. A chromosome region containing CenH3 protein constitutes a single compact domain within a morphologically distinct primary constriction. This region usually spans up to a few Mbp of DNA which is composed mainly of one or few families of centromere-specific satellite DNA. Fabeae tribe of Fabaceae family emerged 16 - 23 million years ago and now comprises ca. 380 species from five genera: Pisum, Lathyrus, Vicia, Lens and Vavilovia. We discovered that Pisum and Lathyrus spp. possess two variants of CenH3, designated as CenH3-1 and CenH3-2, while Lens and Vicia spp. possess just CenH3-2. Phylogenetic analysis revealed that the duplication of the ancestral CenH3 preceded the split of Fabeae species, indicating that the CenH3-1 varia...
Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smal... more Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due ...
In most eukaryotes, centromere is determined by the presence of the centromere-specific histone v... more 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 arran...
Retrotransposons consist of significant portions of many complex eukaryotic genomes and are often... more Retrotransposons consist of significant portions of many complex eukaryotic genomes and are often enriched in heterochromatin. The centromeric retrotransposon (CR) family in grass species is colonized in the centromeres and highly conserved among species that have been diverged for .50 MY. These unique characteristics have inspired scientists to speculate about the roles of CR elements in organization and function of
Continuously growing hairy-root cultures of pea (Pisum sativum L.) line L-84 have been induced by... more Continuously growing hairy-root cultures of pea (Pisum sativum L.) line L-84 have been induced by transformation of seedlings by Agrobacterium rhizogenes A4-24. The cultures were then used for cell cycle synchronisation in vitro and for preparation of chromosome suspensions. Various concentrations of hydroxyurea (0.5–2.0 mM) and timing of amiprophos-methyl (APM, 2–4 h) treatment have been tested to achieve the highest mitotic synchrony, and to accumulate the highest number of synchronised cells in metaphase, respectively. Optimal conditions (2 mM hydroxyurea for 18 h, 10 μM APM for 2 h applied 6 h after a release from hydroxyurea block) resulted in a high frequency of cells in metaphase (43%). The synchronised root tips were fixed in formaldehyde and chromosomes were released into a lysis buffer by mechanical homogenisation. The chromosomes in suspension showed a well preserved morphology, and could be used for flow cytometric analysis. While only two chromosomes (5 and 7) were discriminated in a standard karyotype, four chromosomes (3, 5, 6 and 7) could be clearly discriminated in the line L-84 which contains a stable reciprocal translocation between chromosomes 3 and 6. The establishment of hairy root cultures and the development of an efficient chromosome isolation procedure from a translocation line L-84 represent a basic prerequisite for subsequent flow-sorting of selected chromosomes of pea.
Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structu... more Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structure of their centromeric regions, which is clearly apparent during metaphase by the formation of extended primary constrictions which span up to a third of the length of the chromosome. In addition, these species express two different variants of the CenH3 protein which are co-localized in multiple domains along the poleward surface of the primary constrictions. Here, we show that the constrictions represent a distinct type of chromatin differing from the chromosome arms. In metaphase, histone phosphorylation patterns including H3S10ph, H3S28ph, and H3T3ph were observed along the entire constriction, in a way similar to holocentric chromosomes. On the other hand, distribution of phosphorylated H2AT120 was different from that previously reported from either, holocentric and monocentric chromosomes, occurring at chromatin surrounding but not overlapping CenH3 domains. Since some of these phosphorylations play a role in chromatid cohesion, it can be assumed that they facilitate correct chromosome segregation by ensuring that multiple separate CenH3 domains present on the same chromatid are oriented toward the same pole. The constrictions also displayed distinct patterns of histone methylation marks, being enriched in H3K9me2 and depleted in H3K4me3 and H3K27me2 compared to the chromosome arms. Super-resolution fluorescence microscopy revealed that although both CenH3 protein variants are present in all CenH3 domains detected on metaphase chromosomes, they are only partially co-localized while there are chromatin subdomains which are mostly made of only one CenH3 variant. Taken together, these data revealed specific features of extended primary constrictions of Lathyrus and Pisum and support the idea that they may represent an intermediate stage between monocentric and holocentric chromosomes.
The differential accumulation and elimination of repetitive DNA are key drivers of genome size va... more The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encou...
Proceedings of the National Academy of Sciences, 2015
Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinet... more Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.
The Plant journal : for cell and molecular biology, Jan 20, 2015
Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure... more Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra di...
Centromeres are essential structures for proper chromosome segregation during cell division. Howe... more Centromeres are essential structures for proper chromosome segregation during cell division. However, the relationship between centromere structure, sequence composition and function is still unclear. To study these questions, we adopted a model system of two closely related pea species. Garden pea (Pisum sativum) mitotic chromosomes harbor unique centromeres with multiple functional domains embedded within an extended primary constriction. Also, the sequence composition of the pea centromere differs from any other species studied so far. Pea centromeres are composed of thirteen families of satellite repeats differing in their monomer length. None of the families are present on all chromosomes and individual CenH3 domains on a single chromosome often contain different repeats. Using CenH3 antibody, we detected elongated centromeres also in some of the chromosomes of tawny pea (P. fulvum), a wild relative of the garden pea. To identify repetitive sequences in both genomes, we employe...
Centromeric regions of plant chromosomes are usually made of complex populations of repetitive el... more Centromeric regions of plant chromosomes are usually made of complex populations of repetitive elements which are difficult to investigate using conventional cloning and sequencing approaches. In addition, requirements for large-scale sequencing and the lack of suitable bioinformatics tools have long hampered centromere investigation in non-model species. In this lecture, we will present an experimental workflow that takes advantage of recently introduced next generation sequencing technologies and newly developed computational tools to overcome these limitations and allow investigation of centromere composition in a wide range of plant species. Combining whole genome and CenH3-ChIP sequencing with data analysis using RepeatExplorer pipeline led to the identification of all major centromeric repeats in cultivated pea (Pisum sativum) and its wild relative P. fulvum. Both species have extended centromeres which are composed of multiple CenH3 domains associated with more than ten diffe...
The centromere is a functional chromosome domain that is essential for faithful chromosome segreg... more The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division. It can be reliably identified by the presence of CenH3 histone. In most diploid plant genomes, CenH3 is encoded by a single copy gene. A chromosome region containing CenH3 protein constitutes a single compact domain within a morphologically distinct primary constriction. This region usually spans up to a few Mbp of DNA which is composed mainly of one or few families of centromere-specific satellite DNA. Fabeae tribe of Fabaceae family emerged 16 - 23 million years ago and now comprises ca. 380 species from five genera: Pisum, Lathyrus, Vicia, Lens and Vavilovia. We discovered that Pisum and Lathyrus spp. possess two variants of CenH3, designated as CenH3-1 and CenH3-2, while Lens and Vicia spp. possess just CenH3-2. Phylogenetic analysis revealed that the duplication of the ancestral CenH3 preceded the split of Fabeae species, indicating that the CenH3-1 varia...
Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smal... more Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due ...
In most eukaryotes, centromere is determined by the presence of the centromere-specific histone v... more 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 arran...
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Papers by Pavel Neumann