Reconstructing the evolution of Brachypodium genomes using comparative chromosome painting.

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

Anthropogenic Impacts on Meiosis in Plants

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

Primary group size, social support, gender and future mental health status in a prospective study of people living in private households throughout Great Britain

Background. Structural characteristics of social networks such as primary group size have received less attention than measures of perceived social support. Previous research suggests that associations between social network size and later common mental disorder status may differ according to sex and initial mental state. Method. Adults participating in the 2000 British National Household Survey of psychiatric morbidity were randomly selected for follow-up 18 months later. The revised Clinical Interview Schedule (CIS-R) and the Interview Measure of Social Relations (IMSR) were administered at baseline and follow-up. Primary group size was defined as the total number of close relatives and friends. A four-level scale of common mental disorder was modelled with ordinal logistic regression, based on weighted data (n=2413). Findings. After adjusting for confounders, a primary group size of three or less at time 1 predicted worse mental health at time 2. This effect was greatest in men who were initially non-cases at baseline (averaged odds 4.5) and in women who were initially cases at baseline (average odds 2.9). Primary group size at time 2 was significantly predicted by level of common mental disorder at time 1 in women but not in men. Thus, confounding by baseline disorder does not explain risk of developing poor mental health in socially isolated men. Conclusion. This study replicates the strong effects of primary group size on future mental health that emerge when men and women are studied separately and when subjects are categorized according to baseline mental health status

High Resolution Analysis of Meiotic Chromosome Structure and Behaviour in Barley (Hordeum vulgare L.)

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)

Comparatively Barcoded Chromosomes of Brachypodium Perennials Tell the Story of Their Karyotype Structure and Evolution

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. Brachypodium distachyon 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 di erent 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

CDKG1 protein kinase is essential for synapsis and male meiosis at high ambient temperature in Arabidopsis thaliana

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

B chromosomes are associated with redistribution of genetic recombination towards lower-recombination chromosomal regions in perennial ryegrass

Supernumerary ‘B’ chromosomes are non-essential components of the genome present in a range of plant and animal species – including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT marker genetic maps for 6 perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progenies and their parents we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared to zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, i.e., pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

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

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

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