Helical coiling of metaphase chromatids.

Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.

Defects in meiotic chromosome segregation lead to unreduced male gametes in Arabidopsis SMC5/6 complex mutants.

Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.

Mechanistic transcriptome comprehension of Chlamydomonas reinhardtii subjected to black phosphorus.

Two-dimensional materials have recently gained significant awareness. A representative of such materials, black phosphorous (BP), earned attention based on its comprehensive application potential. The presented study focuses on the mode of cellular response underlying the BP interaction with Chlamydomonas reinhardtii as an algal model organism. We observed noticeable ROS formation and changes in outer cellular topology after 72 h of incubation at 5 mg/L BP. Transcriptome profiling was employed to examine C. reinhardtii response after exposure to 25 mg/L BP for a deeper understanding of the associated processes. The RNA sequencing has revealed a comprehensive response with abundant transcript downregulation. The mode of action was attributed to cell wall disruption, ROS elevation, and chloroplast disturbance. Besides many other dysregulated genes, the cell response involved the downregulation of GH9 and gametolysin within a cell wall, pointing to a shift to discrete manipulation with resources. The response also included altered expression of the PRDA1 gene associated with redox governance in chloroplasts implying ROS disharmony. Altered expression of the Cre-miR906-3p, Cre-miR910, and Cre-miR914 pointed to those as potential markers in stress response studies.

Intact ribosomal DNA arrays of Potentilla origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer.

During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised the possibility of horizontal transfer of a eudicot alien ribosomal DNA (rDNA) into the Erythronium genome. In this work we aimed to support this hypothesis by carrying out genomic, molecular, and cytogenetic analyses. Genome skimming coupled by PacBio HiFi sequencing of a bacterial artificial chromosome clone derived from flow-sorted nuclei was used to characterise the alien 45S rDNA. Integration of alien rDNA in the recipient genome was further proved by Southern blotting and fluorescence in situ hybridization using specific probes. Alien rDNA, nested among Potentilla species in phylogenetic analysis, likely entered the Erythronium lineage in the common ancestor of E. dens-canis and E. caucasicum. Transferred eudicot-type rDNA preserved its tandemly arrayed feature on a single chromosome and was found to be transcribed in the monocot host, albeit much less efficiently than the native counterpart. This study adds a new example to the rarely documented nuclear-to-nuclear jumps of DNA between eudicots and monocots while holding the scientific community continually in suspense about the mode of DNA transfer.

Fine mapping of powdery mildew and stripe rust resistance genes Pm5V/Yr5V transferred from Dasypyrum villosum into wheat without yield penalty.

KEY MESSAGE: The novel wheat powdery mildew and stripe rust resistance genes Pm5V/Yr5V are introgressed from Dasypyrum villosum and fine mapped to a narrowed region in 5VS, and their effects on yield-related traits were characterized. The powdery mildew and stripe rust seriously threaten wheat production worldwide. Dasypyrum villosum (2n = 2x = 14, VV), a relative of wheat, is a valuable resource of resistance genes for wheat improvement. Here, we describe a platform for rapid introgression of the resistance genes from D. villosum into the wheat D genome. A complete set of new wheat-D. villosum V (D) disomic substitution lines and 11 D/V Robertsonian translocation lines are developed and characterized by molecular cytogenetic method. A new T5DL·5V#5S line NAU1908 shows resistance to both powdery mildew and stripe rust, and the resistances associated with 5VS are confirmed to be conferred by seedling resistance gene Pm5V and adult-plant resistance gene Yr5V, respectively. We flow-sort chromosome arm 5VS and sequence it using the Illumina NovaSeq 6000 system that allows us to generate 5VS-specific markers for genetic mapping of Pm5V/Yr5V. Fine mapping shows that Pm5V and Yr5V are closely linked and the location is narrowed to an approximately 0.9 Mb region referencing the sequence of Chinese Spring 5DS. In this region, a NLR gene in scaffold 24,874 of 5VS orthologous to TraesCS5D02G044300 is the most likely candidate gene for Pm5V. Soft- and hard-grained T5DL·5V#5S introgressions confer resistance to both powdery mildew and stripe rust in diverse wheat genetic backgrounds without yield penalty. Meanwhile, significant decrease in plant height and increase in yield were observed in NIL-5DL·5V#5S compared with that in NIL-5DL·5DS. These results indicate that Pm5V/Yr5V lines might have the potential value to facilitate wheat breeding for disease resistance.

Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers.

Crested wheatgrass (Agropyron cristatum), a wild relative of wheat, is an attractive source of genes and alleles for their improvement. Its wider use is hampered by limited knowledge of its complex genome. In this work, individual chromosomes were purified by flow sorting, and DNA shotgun sequencing was performed. The annotation of chromosome-specific sequences characterized the DNA-repeat content and led to the identification of genic sequences. Among them, genic sequences homologous to genes conferring plant disease resistance and involved in plant tolerance to biotic and abiotic stress were identified. Genes belonging to the important groups for breeders involved in different functional categories were found. The analysis of the DNA-repeat content identified a new LTR element, Agrocen, which is enriched in centromeric regions. The colocalization of the element with the centromeric histone H3 variant CENH3 suggested its functional role in the grass centromere. Finally, 159 polymorphic simple-sequence-repeat (SSR) markers were identified, with 72 of them being chromosome- or chromosome-arm-specific, 16 mapping to more than one chromosome, and 71 mapping to all the Agropyron chromosomes. The markers were used to characterize orthologous relationships between A. cristatum and common wheat that will facilitate the introgression breeding of wheat using A. cristatum.

Identification of New QTLs for Dietary Fiber Content in Aegilops biuncialis.

Grain dietary fiber content is an important health-promoting trait of bread wheat. A dominant dietary fiber component of wheat is the cell wall polysaccharide arabinoxylan and the goatgrass Aegilops biuncialis has high ß-glucan content, which makes it an attractive gene source to develop wheat lines with modified fiber composition. In order to support introgression breeding, this work examined genetic variability in grain ß-glucan, pentosan, and protein content in a collection of Ae. biuncialis. A large variation in grain protein and edible fiber content was revealed, reflecting the origin of Ae. biuncialis accessions from different eco-geographical habitats. Association analysis using DArTseq-derived SNPs identified 34 QTLs associated with ß-glucan, pentosan, water-extractable pentosan, and protein content. Mapping the markers to draft chromosome assemblies of diploid progenitors of Ae. biuncialis underlined the role of genes on chromosomes 1Mb, 4Mb, and 5Mb in the formation of grain ß-glucan content, while other QTLs on chromosome groups 3, 6, and 1 identified genes responsible for total- and water-extractable pentosan content. Functional annotation of the associated marker sequences identified fourteen genes, nine of which were identified in other monocots. The QTLs and genes identified in the present work are attractive targets for chromosome-mediated gene transfer to improve the health-promoting properties of wheat-derived foods.

Chromosome analysis and sorting.

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.

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human-Arabidopsis Hybrid Cell Line.

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human-Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.

In-Depth Sequence Analysis of Bread Wheat VRN1 Genes.

The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Multiple displacement amplification of the DNA from single flow-sorted plant chromosome.

A protocol is described for production of micrograms of DNA from single copies of flow-sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome-derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single-chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome-specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole-genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.

Advanced environmental scanning electron microscopy reveals natural surface nano-morphology of condensed mitotic chromosomes in their native state.

The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.

Chromosome Conformation Capture of Mitotic Chromosomes.

Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a method of choice for study of spatial genome-wide interactions. Although its utilization has been focused mainly on studying genomic interactions in interphase nuclei, the method can be also successfully applied to study 3D architecture and genome folding in mitotic chromosomes. However, obtaining sufficient number of mitotic chromosomes as an input material and effective coupling with Hi-C method is challenging in plant species. An elegant way to overcome hindrances with obtaining a pure mitotic chromosome fraction is their isolation via flow cytometric sorting. This chapter presents a protocol describing plant sample preparation for chromosome conformation studies, for flow-sorting of plant mitotic metaphase chromosomes and for the Hi-C procedure.

Flow Sorting-Assisted Optical Mapping.

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

Flow Cytometric Analysis and Sorting of Plant Chromosomes.

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.

The chromatin determinants and Ph1 gene effect at wheat sites with contrasting recombination frequency.

INTRODUCTION: Meiotic recombination is one of the most important processes of evolution and adaptation to environmental conditions. Even though there is substantial knowledge about proteins involved in the process, targeting specific DNA loci by the recombination machinery is not well understood. OBJECTIVES: This study aims to investigate a wheat recombination hotspot (H1) in comparison with a "regular" recombination site (Rec7) on the sequence and epigenetic level in conditions with functional and non-functional Ph1 locus. METHODS: The DNA sequence, methylation pattern, and recombination frequency were analyzed for the H1 and Rec7 in three mapping populations derived by crossing introgressive wheat line 8.1 with cv. Chinese Spring (with Ph1 and ph1 alleles) and cv. Tähti. RESULTS: The H1 and Rec7 loci are 1.586 kb and 2.538 kb long, respectively. High-density mapping allowed to delimit the Rec7 and H1 to 19 and 574 bp and 593 and 571 bp CO sites, respectively. A new method (ddPing) allowed screening recombination frequency in almost 66 thousand gametes. The screening revealed a 5.94-fold higher recombination frequency at the H1 compared to the Rec7. The H1 was also found out of the Ph1 control, similarly as gamete distortion. The recombination was strongly affected by larger genomic rearrangements but not by the SNP proximity. Moreover, chromatin markers for open chromatin and DNA hypomethylation were found associated with crossover occurrence except for the CHH methylation. CONCLUSION: Our results, for the first time, allowed study of wheat recombination directly on sequence, shed new light on chromatin landmarks associated with particular recombination sites, and deepened knowledge about role of the Ph1 locus in control of wheat recombination processes. The results are suggesting more than one recombination control pathway. Understanding this phenomenon may become a base for more efficient wheat genome manipulation, gene pool enrichment, breeding, and study processes of recombination itself.

New insight into the biocompatibility/toxicity of graphene oxides and their reduced forms on Chlamydomonas reinhardtii.

Graphene oxides (GOs) and their reduced forms are often discussed both positively and negatively due to the lack of information about their chemistry and structure. This study utilized GOs with two sheet sizes that were further reduced by two reducing agents (sodium borohydride and hydrazine) to obtain two different degrees of reduction. The synthesized nanomaterials were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), elemental analysis (EA), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy (RA) to understand their chemistry and structure. The second focus of our investigation included in vitro testing of the biocompatibility/toxicity of these materials on a model organism, the freshwater microalga Chlamydomonas reinhardtii. The effects were studied on the basis of biological endpoints complemented by biomass investigation (FTIR spectroscopy, EA, and atomic absorption spectrometry (AAS)). The results showed that the biocompatibility/toxicity of GOs is dependent on their chemistry and structure and that it is impossible to generalize the toxicity of graphene-based nanomaterials.

Transcriptomic hallmarks of in vitro TiO2 nanotubes toxicity in Chlamydomonas reinhardtii.

Recently, more accessible transcriptomic approaches have provided a new and deeper understanding of environmental toxicity. The present study focuses on the transcriptomic profiles of green microalgae Chlamydomonas reinhardtii exposed to new industrially promising material, TiO2 nanotubes (NTs), as an example of a widely used one-dimensional nanomaterial. The first algal in vitro assay included 2.5 and 7.5 mg/L TiO2 NTs, resulting in a dose-dependent negative effect on biological endpoints. At a working concentration of 7.5 mg/L, RNA-sequencing showed a mainly negative effect on the cells. In summary, the results indicated metabolic disruption, such as ATP loss, damage to mitochondria and chloroplasts, loss of solutes due to permeated membranes, and cell wall damage. Moreover, apoptosis-induced transcripts were detected. Interestingly, reactivation of transposons was observed. In signalling and transcription pathways, including chromatin remodelling and locking, the annotated genes were downregulated.