Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

The coordination between nuclear and organellar genes is essential to many aspects of eukaryotic life, including basic metabolism, energy production, and ultimately, organismal fitness. Although nuclear genes are biparentally inherited, mitochondrial and chloroplast genes are almost exclusively maternally inherited, and this asymmetry may lead to a bias in the chromosomal distribution of nuclear genes whose products act in the mitochondria or chloroplasts. In particular, because X-linked genes have a higher probability of cotransmission with organellar genes (2/3) compared with autosomal genes (1/2), selection for coadaptation has been predicted to lead to an overrepresentation of nuclear-mitochondrial and nuclear-chloroplast genes on the X chromosome relative to autosomes. In contrast, the occurrence of sexually antagonistic organellar mutations might lead to selection for movement of cytonuclear genes from the X chromosome to autosomes to reduce male mutation load. Recent broad-scale comparative studies of N-mt distributions in animals have found evidence for these hypotheses in some species, but not others. Here, we use transcriptome sequences to conduct the first study of the chromosomal distribution of cytonuclear interacting genes in a plant species with sex chromosomes (Rumex hastatulus; Polygonaceae). We found no evidence of under- or overrepresentation of either N-mt or N-cp genes on the X chromosome, and thus no support for either the coadaptation or the sexual-conflict hypothesis. We discuss how our results from a species with recently evolved sex chromosomes fit into an emerging picture of the evolutionary forces governing the chromosomal distribution of nuclear-mitochondrial and nuclear-chloroplast genes.

Most flowering plants are hermaphrodites, but around 6% of species are dioecious, having separate male and female plants. Sex chromosomes and some sex-specific genes have been reported in plants, but the genome sequences have not been compared. We now report the genome sequence of male and female jojoba (Simmondsia chinensis) plants, revealing a very large difference in the sex chromosomes. The male genome assembly was 832 Mb and the female 822 Mb. This was explained by the large size differences in the Y chromosome (37.6 Mb) compared with the X chromosome (26.9 Mb). Relative to the X chromosome, the Y chromosome had two large insertions each of more than 5 Mb containing more than 400 genes. Many of the genes in the chromosome-specific regions were novel. These male-specific regions included many flowering-related and stress response genes. Smaller insertions found only in the X chromosome totalled 877 kb. The wide divergence of the sex chromosomes suggests a long period of adaptation to diverging sex-specific roles. Male and female plants may have evolved to accommodate factors such as differing reproductive resource allocation requirements under the stress of the desert environment in which the plants are found. The sex-determining regions accumulate genes beneficial to each sex. This has required the evolution of many more novel sex-specific genes than has been reported for other organisms. This suggest that dioecious plants provide a novel source of genes for manipulation of reproductive performance and environmental adaptation in crops.

In the last decade, progress has been made in methods to identify the sex determination system in plants. This gives the opportunity to study sex chromosomes that arose independently at different phylogenetic scales, and thus allows the discovery and the understanding of early stages of sex chromosome evolution. In the genus Silene, sex chromosomes have evolved independently in at least two clades from a nondioecious ancestor, the Melandrium and Otites sections. In the latter, sex chromosomes could be younger than in the section Melandrium, based on phylogenetic studies and as no heteromorphic sex chromosomes have been detected. This section might also exhibit lability in sex determination, because male heterogamy and female heterogamy have been suggested to occur.In this study, we investigated the sex determination system of two dioecious species in the section Otites (Silene otites and its close relative Silene pseudotites). Applying the new probabilistic method SEX-DETector on RNA-seq data from cross-controlled progenies, we inferred their most likely sex determination system and a list of putative autosomal and sex-linked contigs. We showed that the two phylogenetically close species differed in their sex determination system (XY versus ZW) with sex chromosomes that derived from two different pairs of autosomes. We built a genetic map of the sex chromosomes and showed that both pairs exhibited a large region with lack of recombination. However, the sex-limited chromosomes exhibited no strong degeneration. Finally, using the "ancestral" autosomal expression of sex-linked orthologs of nondioecious S. nutans, we found a slight signature of dosage compensation in the heterogametic females of S. otites.

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.

Genome size evolution is a complex process influenced by polyploidization, satellite DNA accumulation, and expansion of retroelements. How this process could be affected by different reproductive strategies is still poorly understood.

Rumex acetosa is a dioecious plant with the XY1Y2 sex chromosome system. Both Y chromosomes are heterochromatic and are thought to be degenerated. We performed low-pass 454 sequencing and similarity-based clustering of male and female genomic 454 reads to identify and characterize major groups of R. acetosa repetitive DNA. We found that Copia and Gypsy retrotransposons dominated, followed by DNA transposons and nonlong terminal repeat retrotransposons. CRM and Tat/Ogre retrotransposons dominated the Gypsy superfamily, whereas Maximus/Sireviruses were most abundant among Copia retrotransposons. Only one Gypsy subfamily had accumulated on Y1 and Y2 chromosomes, whereas many retrotransposons were ubiquitous on autosomes and the X chromosome, but absent on Y1 and Y2 chromosomes, and others were depleted from the X chromosome. One group of CRM Gypsy was specifically localized to centromeres. We also found that majority of previously described satellites (RAYSI, RAYSII, RAYSIII, and RAE180) are accumulated on the Y chromosomes where we identified Y chromosome-specific variant of RAE180. We discovered two novel satellites-RA160 satellite dominating on the X chromosome and RA690 localized mostly on the Y1 chromosome. The expression pattern obtained from Illumina RNA sequencing showed that the expression of transposable elements is similar in leaves of both sexes and that satellites are also expressed. Contrasting patterns of transposable elements (TEs) and satellite localization on sex chromosomes in R. acetosa, where not only accumulation but also depletion of repetitive DNA was observed, suggest that a plethora of evolutionary processes can shape sex chromosomes.

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.

In various eukaryotes, supernumerary B chromosomes (Bs) are an optional genomic component that affect their integrity and functioning. In the present study, the impact of Bs on the current changes in the genome of goatgrass, Aegilops speltoides, was addressed. Individual plants from contrasting populations with and without Bs were explored using fluorescence in situ hybridization. In parallel, abundances of the Ty1-copia, Ty3-gypsy, and LINE retrotransposons (TEs), and the species-specific Spelt1 tandem repeat (TR) in vegetative and generative spike tissues were estimated by real-time quantitative PCR. The results revealed: (i) ectopic associations between Bs and the regular A chromosomes, and (ii) cell-specific rearrangements of Bs in both mitosis and microgametogenesis. Further, the copy numbers of TEs and TR varied significantly between (iii) genotypes and (iv) different spike tissues in the same plant(s). Finally, (v) in plants with and without Bs from different populations, genomic abundances and/or copy number dynamics of TEs and TR were similar. These findings indicate that fluctuations in TE and TR copy numbers are associated with DNA damage and repair processes during cell proliferation and differentiation, and ectopic recombination is one of the mechanisms by which Bs play a role in genome changes.

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes.

Dioecious plants vary in whether their sex chromosomes are heteromorphic or homomorphic, but even homomorphic sex chromosomes may show divergence between homologues in the non-recombining, sex-determining region (SDR). Very little is known about the SDR of these species, which might represent particularly early stages of sex-chromosome evolution. Here, we assess the size and content of the SDR of the diploid dioecious herb Mercurialis annua, a species with homomorphic sex chromosomes and mild Y-chromosome degeneration. We used RNA sequencing (RNAseq) to identify new Y-linked markers for M. annua. Twelve of 24 transcripts showing male-specific expression in a previous experiment could be amplified by polymerase chain reaction (PCR) only from males, and are thus likely to be Y-linked. Analysis of genome-capture data from multiple populations of M. annua pointed to an additional six male-limited (and thus Y-linked) sequences. We used these markers to identify and sequence 17 sex-linked bacterial artificial chromosomes (BACs), which form 11 groups of non-overlapping sequences, covering a total sequence length of about 1.5 Mb. Content analysis of this region suggests that it is enriched for repeats, has low gene density, and contains few candidate sex-determining genes. The BACs map to a subset of the sex-linked region of the genetic map, which we estimate to be at least 14.5 Mb. This is substantially larger than estimates for other dioecious plants with homomorphic sex chromosomes, both in absolute terms and relative to their genome sizes. Our data provide a rare, high-resolution view of the homomorphic Y chromosome of a dioecious plant.

During mitosis in higher eukaryotes, each chromosome condenses into a pair of rod-shaped chromatids. This process is co-regulated by the activity of several gene families, and the underlying biophysics remains poorly understood. To better understand the factors regulating chromosome condensation, we compiled a database of mitotic chromosome size and DNA content from the tables and figures of >200 published papers. A comparison across vertebrate species shows that chromosome width, length and volume scale with DNA content to the powers ∼1/4, ∼1/2, and ∼1, respectively. Angiosperms (flowering plants) show a similar length scaling, so this result is not specific to vertebrates. Chromosome shape and size thus satisfy two conditions: (1) DNA content per unit volume is approximately constant and (2) the cross-sectional area increases proportionately with chromosome length. Since viscous drag forces during chromosome movement are expected to scale with length, we hypothesize that the cross-section increase is necessary to limit the occurrence of large chromosome elongations that could slow or stall mitosis. Lastly, we note that individual vertebrate karyotypes typically exhibit a wider range of chromosome lengths as compared with angiosperms.

Cohesin is a multi-unit protein complex from the structural maintenance of chromosomes (SMC) family, required for holding sister chromatids together during mitosis and meiosis. In yeast, the cohesin complex entraps sister DNAs within tripartite rings created by pairwise interactions between the central ring units SMC1 and SMC3 and subunits such as the α-kleisin SCC1 (REC8/SYN1 in meiosis). The complex is an indispensable regulator of meiotic recombination in eukaryotes. In Arabidopsis and maize, the SMC1/SMC3 heterodimer is a key determinant of meiosis. In Arabidopsis, several kleisin proteins are also essential: SYN1/REC8 is meiosis-specific and is essential for double-strand break repair, whereas AtSCC2 is a subunit of the cohesin SCC2/SCC4 loading complex that is important for synapsis and segregation. Other important meiotic subunits are the cohesin EXTRA SPINDLE POLES (AESP1) separase, the acetylase ESTABLISHMENT OF COHESION 1/CHROMOSOME TRANSMISSION FIDELITY 7 (ECO1/CTF7), the cohesion release factor WINGS APART-LIKE PROTEIN 1 (WAPL) in Arabidopsis (AtWAPL1/AtWAPL2), and the WAPL antagonist AtSWITCH1/DYAD (AtSWI1). Other important complexes are the SMC5/SMC6 complex, which is required for homologous DNA recombination during the S-phase and for proper meiotic synapsis, and the condensin complexes, featuring SMC2/SMC4 that regulate proper clustering of rDNA arrays during interphase. Meiotic recombination is the key to enrich desirable traits in commercial plant breeding. In this review, I highlight critical advances in understanding plant chromatid cohesion in the model plant Arabidopsis and crop plants and suggest how manipulation of crossover formation during meiosis, somatic DNA repair and chromosome folding may facilitate transmission of desirable alleles, tolerance to radiation, and enhanced transcription of alleles that regulate sexual development. I hope that these findings highlight opportunities for crop breeding.

Coconut is an important tropical oil and fruit crop whose evolutionary position renders it a fantastic species for the investigation of the evolution of monocot chromosomes and the subsequent differentiation of ancient plants.

Genomes undergo changes in organization as a result of gene duplications, chromosomal rearrangements and local mutations, among other mechanisms. In contrast to prokaryotes, in which genes of a common function are often organized in operons and reside contiguously along the genome, most eukaryotes show much weaker clustering of genes by function, except for few concrete functional groups. We set out to check systematically if there is a relation between gene function and gene organization in the human genome. We test this question for three types of functional groups: pairs of interacting proteins, complexes and pathways. We find a significant concentration of functional groups both in terms of their distance within the same chromosome and in terms of their dispersal over several chromosomes. Moreover, using Hi-C contact map of the tendency of chromosomal segments to appear close in the 3D space of the nucleus, we show that members of the same functional group that reside on distinct chromosomes tend to co-localize in space. The result holds for all three types of functional groups that we tested. Hence, the human genome shows substantial concentration of functional groups within chromosomes and across chromosomes in space.

The genomes of many filamentous fungi consist of a 'core' part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage-specific (LS) chromosomes. In the plant-pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the 'effector' LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1-Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole-genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring telocentric chromosomes (telosomes) with a terminal centromere are rare but do exist. Telosomes arise through misdivision of centromeres in normal chromosomes, and their cytological stability depends on the structure of their kinetochores. The instability of telosomes may be attributed to the relative centromere size and the degree of completeness of their kinetochore. Here we test this hypothesis by analyzing the cytogenetic structure of wheat telosomes. We used a population of 80 telosomes arising from the misdivision of the 21 chromosomes of wheat that have shown stable inheritance over many generations. We analyzed centromere size by probing with the centromere-specific histone H3 variant, CENH3. Comparing the signal intensity for CENH3 between the intact chromosome and derived telosomes showed that the telosomes had approximately half the signal intensity compared to that of normal chromosomes. Immunofluorescence of CENH3 in a wheat stock with 28 telosomes revealed that none of the telosomes received a complete CENH3 domain. Some of the telosomes lacked centromere specific retrotransposons of wheat in the CENH3 domain, indicating that the stability of telosomes depends on the presence of CENH3 chromatin and not on the presence of CRW repeats. In addition to providing evidence for centromere shift, we also observed chromosomal aberrations including inversions and deletions in the short arm telosomes of double ditelosomic 1D and 6D stocks. The role of centromere-flanking, pericentromeric heterochromatin in mitosis is discussed with respect to genome/chromosome integrity.

The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biological processes, such as DNA replication, transcription, and repair. However, detailed data about the CT arrangement in monocotyledonous plants are scarce. In this study, chromosome painting was used to analyse the distribution and associations of individual chromosomes in the 3-D preserved nuclei of Brachypodium distachyon root cells in order to determine the factors that may have an impact on the homologous CT arrangement. It was shown that the frequency of CT association is linked to the steric constraints imposed by the limited space within the nucleus and may depend on chromosome size and morphology as well as on the nuclear shape. Furthermore, in order to assess whether the distribution of interphase chromosomes is random or is subject to certain patterns, a comparison between the experimental data and the results of a computer simulation (ChroTeMo), which was based on a fully probabilistic distribution of the CTs, was performed. This comparison revealed that homologous chromosome arm CTs associate more often than if they were randomly arranged inside the interphase nucleus.