Glyn Jenkins

Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization

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.

Evolution and taxonomic split of the model grass Brachypodium distachyon

BACKGROUND AND AIMS Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n = 10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species. METHODS Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated. KEY RESULTS The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species. CONCLUSIONS The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.

Exploiting the Brachypodium Tool Box in cereal and grass research

It is now a decade since Brachypodium distachyon (Brachypodium) was suggested as a model species for temperate grasses and cereals. Since then transformation protocols, large expressed sequence tag (EST) databases, tools for forward and reverse genetic screens, highly refined cytogenetic probes, germplasm collections and, recently, a complete genome sequence have been generated. In this review, we will describe the current status of the Brachypodium Tool Box and how it is beginning to be applied to study a range of biological traits. Further, as genomic analysis of larger cereals and forage grasses genomes are becoming easier, we will re-evaluate Brachypodium as a model species. We suggest that there remains an urgent need to employ reverse genetic and functional genomic approaches to identify the functionality of key genetic elements, which could be employed subsequently in plant breeding programmes; and a requirement for a Pooideae reference genome to aid assembling large pooid genomes. Brachypodium is an ideal system for functional genomic studies, because of its easy growth requirements, small physical stature, and rapid life cycle, coupled with the resources offered by the Brachypodium Tool Box.

Ribosomal DNA is an effective marker of Brassica chromosomes

Abstract  Simultaneous fluorescence in situ hybridisation with 5S and 25S rDNA probes enables the discrimination of a substantial number of chromosomes of the complement of all diploid and tetraploid Brassica species of the ”U-triangle”, and provides new chromosomal landmarks for the identification of some chromosomes of this genus which were hitherto indistinguishable. Twelve out of 20 chromosomes can be easily identified in diploid Brassica campestris (AA genome), eight out of 16 in Brassica nigra (BB genome), and six out of 18 in Brassica oleracea (CC genome). Furthermore, just two rDNA markers permit 20 out of 36 chromosomes to be distinguished and assigned to either the A or B genomes of the allotetraploid Brassica juncea, and 18 out of 38 chromosomes identified and assigned to the A or C genomes of the allotetraploid Brassica napus. The number of chromosomes bearing rDNA sites in the tetraploids is not in all cases simply the sum of the numbers of sites in their diploid ancestors. This observation is discussed in terms of the phylogeny and variability within the genomes of the species of this group.

Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv.

Brachypodium distachyon is a ubiquitous, temperate grass species which is being developed and exploited as an alternative model to rice, in order to gain access to important syntenic regions of the genomes of less tractable relatives such as wheat. As part of this initiative, this paper describes for the first time the cytotaxonomy of members of the polyploid series of this species, and challenges the assumption that the series evolved simply by chromosome doubling. In situ hybridization using genomic DNA probes and rDNA markers uncovers a hybrid origin of several of the polyploid ecotypes, and sheds light upon the complex evolution of this species and its close relatives.

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification

BackgroundRed 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.ResultsPlants 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 de 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.ConclusionsThis de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted 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.

Synaptonemal complex formation in hybrids of Lolium temulentum x Lolium perenne (L.) : III. Tetraploid

Comparisons were made between two kinds of tetraploids derived from the hybrid Lolium temulentum x L. perenne. One hybrid behaves like an autotetraploid with multivalents at first metaphase of meiosis in pollen mother cells. The other behaves like an allotetraploid, in which pairing at first metaphase is restricted to bivalents comprised of strictly homologous chromosomes. The “diploidisation” of the latter form is controlled by determinants located on both the normal, A chromosomes and on supernumary B chromosomes. Reconstruction of synaptonemal complexes and their elements, from serial sections through pollen mother cell nuclei examined under the electron microscope, reveals that at zygotene pairing in both forms results in multivalent formation involving non-homologous as well as homologous chromosomes. The mechanism responsible for the diploidisation is, therefore, not based on a restriction of pairing at early meiosis to homologous chromosomes but on a “correction” or transformation of the multivalent chromosome associations to bivalents subsequent to zygotene. The transformation is not completed until late pachytene. In the multivalent-forming tetraploid a maximum of four chromosomes are associated at first metaphase. Yet configurations of a higher valency are found at zygotene. There is, therefore, a partial transformation of multivalents even in this autotetraploid form which restricts configurations at metaphase I to homologous and homoeologous chromosomes only. In both hybrids some homologous bivalents are not the product of resolution of multivalents but result from two-by-two pairing from the beginning of zygotene.

Painting the chromosomes of Brachypodium—current status and future prospects

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.

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).

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.