Christopher Roberts Supervisor: Dr. Luis Mur (former IBS) and Dr. Ian Armstead (formerly IGER) Institute of Biological, Environmental and Rural Sciences, Aberystwyth University Introduction In spite of evolutionary divergence and the pressures of domestication, there’s been a noticeable conservation of genetic synteny between related plant species. From the angles of both evolutionary genetics and plant breeding, there’s been considerable interest in defining these interrelationships. Comparative studies will continue to be of great use in transferring information from the model species to the crop species, as physically ordered complete genome sequence are only available for rice (Oryza sativa) and Arabidopsis thaliana, both of which have relatively small genomes. Using rice as an anchor, DNA sequences from both crop and model species can be aligned with the annotated rice pseudomolecules, creating heat maps (Armstead et al 2007) ‘Hot spots’ within the genomes may be revealed where gene sequences are well or poorly conserved between species, making it possible to establish an overall picture of gene similarities, and observe where genes with differing levels of conservation are clustered. The wild grass species Brachypodium distachyon 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. It has a small genome (380 Mbp), low degree of repetitive DNA, availability of diploid ecotypes, self fertility promoting inbreeding, transformability, small stature, and a moderately rapid life cycle. (Garvin et al 2008) The aim of this project was to compare the genomes of Rice and Brachapodium, developing heat maps based on array intensity scores obtained from Rice Affymetrix microarrays probed with genomic DNA from Brachypodium accessions Bd21 and Bd3-1, produced as part of their on going Brachypodium project by the Mur Group collaborated with Nottingham Arabidopsis Stock Centre (NASC). Materials and Method Sequence alignments were performed using standalone MegaBLAST obtained from NCBI, BLAST FTP site (ftp://ftp.ncbi.nih.gov/blast/executables/release/) with default settings except for a window size of 16 (-W 16), maximum expectation value of 1 × (-e 1e-010) and alignment output (-D 3). Colour coded moving windows (MWs) of 100 genes along each pseudomolecule where used (in Excel) to generate the ‘heat maps’ consisting of 10 colours (blue<red) with each colour representing 10% (or as close to as the data allowed) of the MWs ranked according to array intensity scores Results and Discussion Figure 1 shows the two Brachypodium accessions are very similar. The ‘hot’ (red) and ‘cold’ (blue) spots relate to relative numbers of significant alignments. Hot spots may represent regions in the rice genome which are more conserved within Brachypodium and have more generalised functions, while cold spots may represent regions with more rice specific functions and more rapid evolution so they are less conserved between the species. Cold spots suggest a greater degree of DNA coding sequence differences and so possible divergence in structure and function of the protein products from those regions in the genome. The conserved blocks are not huge showing there are not large duplications or translocations, suggesting Brachypodium may not be as good a model/bridge species as originally proposed. Differences between the 2 Brachypodium accessions where then observed and explored in more detail looking at blocks that had particularly high (red) or low (blue) array intensities, to see if analysis of the annotations of rice genes from ‘hot spots’ and ‘cold spots’ within the heat maps indicated possible functional differences. Figure 2 and 3 show one such region This is a hot spot (high array intensity) on pseudomolecule 3. A large proportion was contributed by transposons/retrotransposons, proteins with catalytic activity, hypothetical proteins, and expressed proteins. Figure 3 shows that when this red region of BD1 was compared with the same region in the image where each individual loci is colour coded by array intensity (figure 1) it can be seen that 11 of the loci have scores assigned the red colour, with the other values ranging right through to dark blue accounting for 9 of the loci. There are differences between the images in figure 1 and 2 because with the moving windows individual high scores can have a disproportionate effect on the overall average Clearly these families of proteins are annotated on the basis of the presence of particular structural motifs in the proteins, rather than by well developed knowledge of their precise functions. There is also likely to be a degree of overlap between members of some of these families functionally. Fold difference was used to show the differences between the two Brachypodium accessions with traditional microarray colours, highlighting the differences on an individual loci basis, which can be viewed in Figure 4. Figure 5 shows a moving widows image generated for bd1 only, with the loci coding for expressed proteins, hypothetical proteins and transposons removed next to the original bd1 MW image. This shows that when the loci with these most numerous annotations are removed, regions are still conserved, most markably at the end of pseudomolecule 10, but the pseudomolecules reduce in size by greater than 50% Figure 1 Heat maps for array intensity Colour coded moving windows used to score array intensity for each of the pseudomolecules (1-12) for both accession bd1 and bd3 Figure 2 Investigation of red region on pseudomolecule 3 Table of the frequency of putative functions of a red section of bd1, together with pie chart Figure 3 Further investigation of red region on pseudomolecule 3 Figure 4 Array intensity 2 fold difference between bd1 and bd3 Red indicates loci with a fold difference below 0.5. Black indicates loci with a fold difference between 0.5 and 2.0. Green indicates loci with a fold difference greater than 2.0. Green therefore highlights loci that are found more in BD1, while red allows us to visualise loci present in BD3. Figure 5 Bd1 Heat maps for array intensity Colour coded moving windows used to score array intensity for each of the pseudomolecules (1-12) for accession bd1. For each rice pseudomolecule column 1 contains all the loci. Column 2 has had the loci coding for expressed proteins, hypothetical proteins and transposons removed References DAVID F. GARVIN, YONG-QIANG GU, ROBERT HASTEROK, SAMUEL P. HAZEN, GLYN JENKINS, TODD C. MOCKLER, LUIS A. J. MUR, AND JOHN P. VOGEL (2008) Development of Genetic and Genomic Research Resources for Brachypodium distachyon, a New Model System for Grass Crop Research. The plant Genome (A Supplement to Crop Science) IAN ARMSTEAD, LIN HUANG, JULIE KING, HELEN OUGHAM, HOWARD THOMAS AND IAN KING (2007) Rice pseudomolecule- anchored cross species DNA sequence alignments indicate regional genomic variation in expressed sequence conservation. BMC Genomics