Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

• Keywords
• CENH3, Centromere, KNL1, NDC80, immunolabeling, kinetochore,
• MeSH
• Centromere * MeSH
• Chromatin MeSH
• Kinetochores * MeSH
• Plant Proteins * genetics   MeSH
• Chromosome Segregation MeSH
• Amino Acid Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Plant Proteins * MeSH

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

• Keywords
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• Chromosomes, Plant * MeSH
• Chromosomes * MeSH
• Cytogenetics MeSH
• Karyotyping MeSH
• Flow Cytometry methods   MeSH
• Suspensions MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Suspensions MeSH

Centromeres in the legume genera Pisum and Lathyrus exhibit unique morphological characteristics, including extended primary constrictions and multiple separate domains of centromeric chromatin. These so-called metapolycentromeres resemble an intermediate form between monocentric and holocentric types, and therefore provide a great opportunity for studying the transitions between different types of centromere organizations. However, because of the exceedingly large and highly repetitive nature of metapolycentromeres, highly contiguous assemblies needed for these studies are lacking. Here, we report on the assembly and analysis of a 177.6 Mb region of pea (Pisum sativum) chromosome 6, including the 81.6 Mb centromere region (CEN6) and adjacent chromosome arms. Genes, DNA methylation profiles, and most of the repeats were uniformly distributed within the centromere, and their densities in CEN6 and chromosome arms were similar. The exception was an accumulation of satellite DNA in CEN6, where it formed multiple arrays up to 2 Mb in length. Centromeric chromatin, characterized by the presence of the CENH3 protein, was predominantly associated with arrays of three different satellite repeats; however, five other satellites present in CEN6 lacked CENH3. The presence of CENH3 chromatin was found to determine the spatial distribution of the respective satellites during the cell cycle. Finally, oligo-FISH painting experiments, performed using probes specifically designed to label the genomic regions corresponding to CEN6 in Pisum, Lathyrus, and Vicia species, revealed that metapolycentromeres evolved via the expansion of centromeric chromatin into neighboring chromosomal regions and the accumulation of novel satellite repeats. However, in some of these species, centromere evolution also involved chromosomal translocations and centromere repositioning.

• MeSH
• Centromere genetics   MeSH
• Chromatin genetics   MeSH
• Pisum sativum * genetics   MeSH
• Humans MeSH
• Chromosomes, Human, Pair 6 * MeSH
• DNA, Satellite genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA, Satellite MeSH

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

• Keywords
• DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
• MeSH
• Cell Cycle MeSH
• Chromosomes, Plant * genetics   MeSH
• Metaphase MeSH
• Flow Cytometry MeSH
• Plants * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Satellite repeats are major sequence constituents of centromeres in many plant and animal species. Within a species, a single family of satellite sequences typically occupies centromeres of all chromosomes and is absent from other parts of the genome. Due to their common origin, sequence similarities exist among the centromere-specific satellites in related species. Here, we report a remarkably different pattern of centromere evolution in the plant tribe Fabeae, which includes genera Pisum, Lathyrus, Vicia, and Lens. By immunoprecipitation of centromeric chromatin with CENH3 antibodies, we identified and characterized a large and diverse set of 64 families of centromeric satellites in 14 species. These families differed in their nucleotide sequence, monomer length (33-2,979 bp), and abundance in individual species. Most families were species-specific, and most species possessed multiple (2-12) satellites in their centromeres. Some of the repeats that were shared by several species exhibited promiscuous patterns of centromere association, being located within CENH3 chromatin in some species, but apart from the centromeres in others. Moreover, FISH experiments revealed that the same family could assume centromeric and noncentromeric positions even within a single species. Taken together, these findings suggest that Fabeae centromeres are not shaped by the coevolution of a single centromeric satellite with its interacting CENH3 proteins, as proposed by the centromere drive model. This conclusion is also supported by the absence of pervasive adaptive evolution of CENH3 sequences retrieved from Fabeae species.

• Keywords
• CENH3, ChIP-seq, centromere evolution, plant chromosomes, satellite DNA,
• MeSH
• Centromere chemistry   MeSH
• Species Specificity MeSH
• Fabaceae genetics   MeSH
• Genetic Variation * MeSH
• DNA, Satellite chemistry   MeSH
• Selection, Genetic MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DNA, Satellite MeSH

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel's original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

• MeSH
• Chromosomes, Plant genetics   MeSH
• Fabaceae classification   genetics   MeSH
• Phenotype MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genome, Plant * MeSH
• Genomics MeSH
• Pisum sativum genetics   MeSH
• Quantitative Trait Loci * MeSH
• Chromosome Mapping MeSH
• Evolution, Molecular * MeSH
• Reference Standards MeSH
• Gene Expression Regulation, Plant MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Plant Proteins genetics   MeSH
• Whole Genome Sequencing MeSH
• Seed Storage Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Plant Proteins MeSH
• Seed Storage Proteins MeSH

The centromere is the region on a chromosome where the kinetochore assembles and spindle microtubules attach during mitosis and meiosis. In the vast majority of eukaryotes, the centromere position is determined epigenetically by the presence of the centromere-specific histone H3 variant CENH3. In species with monocentric chromosomes, CENH3 is confined to a single chromosomal region corresponding to the primary constriction on metaphase chromosomes. By contrast, in holocentrics, CENH3 (and thus centromere activity) is distributed along the entire chromosome length. Here, we report a unique pattern of CENH3 distribution in the holocentric plant Cuscuta europaea. This species expressed two major variants of CENH3, both of which were deposited into one to three discrete regions per chromosome, whereas the rest of the chromatin appeared to be devoid of CENH3. The two CENH3 variants fully co-localized, and their immunodetection signals overlapped with the positions of DAPI-positive heterochromatic bands containing the highly amplified satellite repeat CUS-TR24. This CENH3 distribution pattern contrasted with the distribution of the mitotic spindle microtubules, which attached at uniform density along the entire chromosome length. This distribution of spindle attachment sites proves the holocentric nature of C. europaea chromosomes and also suggests that, in this species, CENH3 either lost its function or acts in parallel to an additional CENH3-free mechanism of kinetochore positioning.

• Keywords
• CENH3, Cuscuta, centromere, holocentric chromosomes, kinetochore, repetitive DNA analysis, satellite DNA,
• Publication type
• Journal Article MeSH

A genetic linkage map of dioecious garden asparagus (Asparagus officinalis L., 2n = 2x = 20) was constructed using F1 population, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. In total, 1376 SNPs and 27 SSRs were used for genetic mapping. Two resulting parental maps contained 907 and 678 markers spanning 1947 and 1814 cM, for female and male parent, respectively, over ten linkage groups representing ten haploid chromosomes of the species. With the aim to anchor the ten genetic linkage groups to individual chromosomes and develop a tool to facilitate genome analysis and gene cloning, we have optimized a protocol for flow cytometric chromosome analysis and sorting in asparagus. The analysis of DAPI-stained suspensions of intact mitotic chromosomes by flow cytometry resulted in histograms of relative fluorescence intensity (flow karyotypes) comprising eight major peaks. The analysis of chromosome morphology and localization of 5S and 45S rDNA by FISH on flow-sorted chromosomes, revealed that four chromosomes (IV, V, VI, VIII) could be discriminated and sorted. Seventy-two SSR markers were used to characterize chromosome content of individual peaks on the flow karyotype. Out of them, 27 were included in the genetic linkage map and anchored genetic linkage groups to chromosomes. The sex determining locus was located on LG5, which was associated with peak V representing a chromosome with 5S rDNA locus. The results obtained in this study will support asparagus improvement by facilitating targeted marker development and gene isolation using flow-sorted chromosomes.

• Keywords
• Asparagus officinalis, FISH, SNPs, SSRs, flow-sorted chromosomes, genetic map, sex chromosome,
• Publication type
• Journal Article MeSH

Satellite DNA, a class of repetitive sequences forming long arrays of tandemly repeated units, represents substantial portions of many plant genomes yet remains poorly characterized due to various methodological obstacles. Here we show that the genome of the field bean (Vicia faba, 2n = 12), a long-established model for cytogenetic studies in plants, contains a diverse set of satellite repeats, most of which remained concealed until their present investigation. Using next-generation sequencing combined with novel bioinformatics tools, we reconstructed consensus sequences of 23 novel satellite repeats representing 0.008-2.700% of the genome and mapped their distribution on chromosomes. We found that in addition to typical satellites with monomers hundreds of nucleotides long, V. faba contains a large number of satellite repeats with unusually long monomers (687-2033 bp), which are predominantly localized in pericentromeric regions. Using chromatin immunoprecipitation with CenH3 antibody, we revealed an extraordinary diversity of centromeric satellites, consisting of seven repeats with chromosome-specific distribution. We also found that in spite of their different nucleotide sequences, all centromeric repeats are replicated during mid-S phase, while most other satellites are replicated in the first part of late S phase, followed by a single family of FokI repeats representing the latest replicating chromatin.

• MeSH
• Molecular Sequence Annotation MeSH
• Centromere metabolism   MeSH
• Chromatin Immunoprecipitation MeSH
• DNA, Plant genetics   metabolism   MeSH
• Genome, Plant genetics   MeSH
• Chromosome Mapping methods   MeSH
• Evolution, Molecular MeSH
• DNA Replication Timing genetics   MeSH
• DNA, Satellite genetics   MeSH
• Sequence Analysis, DNA MeSH
• Vicia faba genetics   metabolism   MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Satellite MeSH

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.

• Keywords
• centromere structure, epigenetic modifications, histone methylation, histone phosphorylation, holocentric, meta-polycentric chromosomes,
• Publication type
• Journal Article MeSH