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Gross crop genomics research frequently is hindered by large genome sizes and polyploidy. While rice is an attractive system for grass genomics due to its small genome size and available genome sequence, it is not particularly well-suited as o robust model system for all grass crops. The wild grass species Brachypodium distachyon (L.) P. Beauv. (Brachypodium) has recently gained favor as a new model system for grass crop genomics research because it possesses a suite of biological traits desired in a model system. Further, it is more closely related to the large and diverse group of cool season grass crops than is either rice (Oryza sativa L.) or sorghum [Sorghum bicolor (L.) Moench.], the second grass crop species whose genome has been sequenced. Thus, by virtue both of its biological attributes and its evolutionary history, Brachypodium fills an important gap in grass crop genomics research. A surge in interest in Brachypodium has led to rapid and significant advances in the acquisition of knowledge and development of resources needed to exploit this species as a model system, including, the impending completion of a draft nuclear genome sequence of Brachypodium. Integration of diverse genetic and genomic resources developed or under development for Brochypodium with the genome sequence will encourage further adoption of this species as a bona fide model plant system.

Abstract: White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial [...]

As part of an initiative to develop Brachypodium distachyon as a genomic 'bridge' species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

A lineage of CACTA family transposons has been identified in temperate grasses and cereals, and a full-length representative of the subfamily from Lolium perenne has been sequenced. Both the size andinternal organization of the L. perenne element are typical of other CACTA family elements but its high copy number and strong conservation are unexpected. Comparison with homologs in other species suggests that this lineage has adopted a distinct and novel evolutionary strategy, which has allowed it to maintain its presence in genomes over long periods of time.

Abstract Background A modification of a standard method of fluorescence in situ hybridisation (FISH) is described, by which a combination of several substrates and probes on single microscope slides enables more accurate comparisons of the distribution and abundance of chromosomal sequences and improves the relatively low throughput of standard FISH methods. Results The utility and application of multi-colour, multi-substrate FISH is illustrated by the simultaneous physical mapping of retrotransposon sequences to three species of Avena, and single locus BAC (bacterial artificial chromosome) clones and rDNA probes to three species of Brachypodium, demonstrating how this would enable better understanding of complex phylogenetic relationships among some of the species belonging to these two genera. Conclusion The results show that use of multi-substrate chromosome preparations significantly increases the utility of FISH in comparative analyses of the distribution and abundance of chromosomal sequences in closely related plant species.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum , describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium -mediated transformation and the selection and analysis of regenerated plants. In B. distachyon , we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits. [ABSTRACT FROM AUTHOR]

Retrotransposon or retrotransposon-like sequences have been reported to be conserved components of cereal centromeres. Here we show that the published sequences are derived from a single conventional Ty3-gypsy family or a nonautonomous derivative. Both autonomous and nonautonomous elements are likely to have colonized Poaceae centromeres at the time of a common ancestor but have been maintained since by active retrotransposition. The retrotransposon family is also present at a lower copy number in the Arabidopsis genome, where it shows less pronounced localization. The history of the family in the two types of genome provides an interesting contrast between 'boom and bust' and persistent evolutionary patterns.

The most distinctive region of the rye B chromosome is a subtelomeric domain that contains an exceptional concentration of B-chromosome-specific sequences. At metaphase this domain appears to be the physical counterpart of the subtelomeric heterochromatic regions present on standard rye chromosomes, but its conformation at interphase is less condensed. In this report we show that the two sequence families that have been previously found to make up the bulk of the domain have been assembled from fragments of a variety of sequence elements, giving rise to their ostensibly foreign origin. A single mechanism, probably based on synthesis-dependent strand annealing (SDSA), is responsible for their assembly. We provide evidence for sequential evolution of one family on the B chromosome itself. The extent of these rearrangements and the complexity of the higher-order organization of the B-chromosome-specific families indicate that instability is a property of the domain itself, rather than of any single sequence. Indirect evidence suggests that particular fragments may have been selected to confer different properties on the domain and that rearrangements are frequently selected for their effect on DNA structure. The current organization appears to represent a transient stage in the evolution of a conventional heterochromatic region from complex sequences.

As the human population grows and continues to encroach on the natural environment, organisms that form part of such ecosystems are becoming increasingly exposed to exogenous anthropogenic factors capable of changing their meiotic landscape. Meiotic recombination generates much of the genetic variation in sexually reproducing species and is known to be a highly conserved pathway. Environmental stresses, such as variations in temperature, have long been known to change the pattern of recombination in both model and crop plants, but there are other factors capable of causing genome damage, infertility and meiotic abnormalities. Our agrarian expansion and our increasing usage of agrochemicals unintentionally affect plants via groundwater contamination or spray drift; our industrial developments release heavy metals into the environment; pathogens are spread by climate change and a globally mobile population; imperfect waste treatment plants are unable to remove chemical and pharmaceutical residues from sewage leading to the release of xenobiotics, all with potentially deleterious meiotic effects. In this review, we discuss the major classes of exogenous anthropogenic factors known to affect meiosis in plants, namely environmental stresses, agricultural inputs, heavy metals, pharmaceuticals and pathogens. The possible evolutionary fate of plants thrust into their new anthropogenically imposed environments are also considered. [ABSTRACT FROM AUTHOR]

Barley ( Hordeum vulgare) is a crop of global significance. However, a third of the genes of barley are largely inaccessible to conventional breeding programmes as crossovers are localised to the ends of the chromosomes. This work examines whether crossovers can be shifted to more proximal regions simply by elevating growth temperature., We utilised a genome-wide marker set for linkage analysis combined with cytological mapping of crossover events to examine the recombination landscape of plants grown at different temperatures., We found that barley shows heterochiasmy, that is, differences between female and male recombination frequencies. In addition, we found that elevated temperature significantly changes patterns of recombination in male meiosis only, with a repositioning of Class I crossovers determined by cytological mapping of Hv MLH3 foci. We show that the length of synaptonemal complexes in male meiocytes increases in response to temperature., The results demonstrate that the distribution of crossover events are malleable and can be shifted to proximal regions by altering the growth temperature. The shift in recombination is the result of altering the distribution of Class I crossovers, but the higher recombination at elevated temperatures is potentially not the result of an increase in Class I events. [ABSTRACT FROM AUTHOR]

Brachypodium distachyon is a model for the temperate cereals and grasses and has a biology, genomics infrastructure and cytogenetic platform fit for purpose. It is a member of a genus with fewer than 20 species, which have different genome sizes, basic chromosome numbers and ploidy levels. The phylogeny and interspecific relationships of this group have not to date been resolved by sequence comparisons and karyotypical studies. The aims of this study are not only to reconstruct the evolution of Brachypodium karyotypes to resolve the phylogeny, but also to highlight the mechanisms that shape the evolution of grass genomes. This was achieved through the use of comparative chromosome painting (CCP) which hybridises fluorescent, chromosome-specific probes derived from B. distachyon to homoeologous meiotic chromosomes of its close relatives. The study included five diploids (B. distachyon 2n = 10, B. sylvaticum 2n = 18, B. pinnatum 2n = 16; 2n = 18, B. arbuscula 2n = 18 and B. stacei 2n = 20) three allotetraploids (B. pinnatum 2n = 28, B. phoenicoides 2n = 28 and B. hybridum 2n = 30), and two species of unknown ploidy (B. retusum 2n = 38 and B. mexicanum 2n = 40). On the basis of the patterns of hybridisation and incorporating published data, we propose two alternative, but similar, models of karyotype evolution in the genus Brachypodium. According to the first model, the extant genome of B. distachyon derives from B. mexicanum or B. stacei by several rounds of descending dysploidy, and the other diploids evolve from B. distachyon via ascending dysploidy. The allotetraploids arise by interspecific hybridisation and chromosome doubling between B. distachyon and other diploids. The second model differs from the first insofar as it incorporates an intermediate 2n = 18 species between the B. mexicanum or B. stacei progenitors and the dysploidic B. distachyon. [ABSTRACT FROM AUTHOR]

Background Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A dec novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions This dec novo assembly of a red clover transcriptome from leaf material of droughted and nondroughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover. [ABSTRACT FROM AUTHOR]

The Arabidopsis cyclin-dependent kinase G (CDKG) gene defines a clade of cyclin-dependent protein kinases related to CDK10 and CDK11, as well as to the enigmatic Ph1-related kinases that are implicated in controlling homeologous chromosome pairing in wheat. Here we demonstrate that the CDKG1/CYCLINL complex is essential for synapsis and recombination during male meiosis. A transfer-DNA insertional mutation in the cdkg1 gene leads to a temperature-sensitive failure of meiosis in late Zygotene/Pachytene that is associated with defective formation of the synaptonemal complex, reduced bivalent formation and crossing over, and aneuploid gametes. An aphenotypic insertion in the cyclin L gene, a cognate cyclin for CDKG, strongly enhances the phenotype of cdkg1-1 mutants, indicating that this cdk-cyclin complex is essential for male meiosis. Since CYCLINL, CDKG, and their mammalian homologs have been previously shown to affect mRNA processing, particularly alternative splicing, our observations also suggest a mechanism to explain the widespread phenomenon of thermal sensitivity in male meiosis. [ABSTRACT FROM AUTHOR]

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM). [ABSTRACT FROM AUTHOR]

Chromosome painting is one of the most powerful and spectacular tools of modern molecular cytogenetics, enabling complex analyses of nuclear genome structure and evolution. For many years, this technique was restricted to the study of mammalian chromosomes, as it failed to work in plant genomes due mainly to the presence of large amounts of repetitive DNA common to all the chromosomes of the complement. The availability of ordered, chromosome-specific BAC clones of Arabidopsis thaliana containing relatively little repetitive genomic DNA enabled the first chromosome painting in dicotyledonous plants. Here, we show for the first time chromosome painting in three different cytotypes of a monocotyledonous plant-the model grass, Brachypodium distachyon. Possible directions of further detailed studies are proposed, such as the evolution of grass karyotypes, the behaviour of meiotic chromosomes, and the analysis of chromosome distribution at interphase. [ABSTRACT FROM AUTHOR]

Background: A modification of a standard method of fluorescence in situ hybridisation (FISH) is described, by which a combination of several substrates and probes on single microscope slides enables more accurate comparisons of the distribution and abundance of chromosomal sequences and improves the relatively low throughput of standard FISH methods. Results: The utility and application of multi-colour, multi-substrate FISH is illustrated by the simultaneous physical mapping of retrotransposon sequences to three species of Avena, and single locus BAC (bacterial artificial chromosome) clones and rDNA probes to three species of Brachypodium, demonstrating how this would enable better understanding of complex phylogenetic relationships among some of the species belonging to these two genera. Conclusion: The results show that use of multi-substrate chromosome preparations significantly increases the utility of FISH in comparative analyses of the distribution and abundance of chromosomal sequences in closely related plant species. [ABSTRACT FROM AUTHOR]

The Brachypodium genus is an informative model system for studying grass karyotype organization. Previous studies of a limited number of species and reference chromosomes have not provided a comprehensive picture of the enigmatic phylogenetic relationships in the genus. Comparative chromosome barcoding, which enables the reconstruction of the evolutionary history of individual chromosomes and their segments, allowed us to infer the relationships between putative ancestral karyotypes of extinct species and extant karyotypes of current species. We used over 80 chromosome-specific BAC (bacterial artificial chromosome) clones derived from five reference chromosomes of B. distachyon as probes against the karyotypes of twelve accessions representing five diploid and polyploid Brachypodium perennials. The results showed that descending dysploidy is common in Brachypodium and occurs primarily via nested chromosome fusions. Brachypodiumdistachyon was rejected as a putative ancestor for allotetraploid perennials and B. stacei for B. mexicanum. We propose two alternative models of perennial polyploid evolution involving either the incorporation of a putative x = 5 ancestral karyotype with different descending dysploidy patterns compared to B. distachyon chromosomes or hybridization of two x = 9 ancestors followed by genome doubling and descending dysploidy. Details of the karyotype structure and evolution in several Brachypodium perennials are revealed for the first time. [ABSTRACT FROM AUTHOR]

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery. [ABSTRACT FROM AUTHOR]

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations. [ABSTRACT FROM AUTHOR]