We are contributing towards building a genetically anchored physical map of the 5300Mb barley genome (almost double that of humans). Our efforts are associated with a collaborative project between IPK Gatersleben (IPK), University of California Riverside (UCR) and the Australian Centre for Plant Functional Genomics (ACPFG) that has initiated the process of building a High Information Content BAC Fingerprint based physical map of the barley genome. We aim to anchor these physical BAC contigs to the barley genetic map using funding we have obtained in two projects (BARCODE and Triticeae Genome). These projects build upon a high density barley gene map we have previously constructed in other projects (including the AGOUEB SA LINK project).

The aim is conceptually simple – to identify BAC contigs containing genes that have been mapped onto the barley gene map. As we are focussing specifically on genes, we are able to relate their position on the barley genetic map with the position of their putative orthologues on the rice and Brachypodium ‘model’ genome sequences. This helps us develop more genetic markers (if required) and provides an idea of the likely local gene content around each of the anchored genes. The outputs of this will help the entire community streamline the cloning of genes controlling important traits based on their position on the genetic map and understanding how they work. Using routinely applied techniques we envisage that it would most likely take some years to get close to completing this task, so we are also exploring alternative approaches to streamline the process. We are of course lucky to have some idea of local gene content and order in the shape of the model genome sequences. We are taking the following approaches.

BAC End Sequencing

As part of the BARCODE project (SCRI, IPK and University of Udine) we are sequencing both ends of approximately 300,000 BAC clones (600,000 reads) that have already been fingerprinted as part of the IPK-led effort described above. This process is already well underway. From analysis of the existing data and comparable data from wheat we estimate that 1-2% of the end sequences will represent genes. If it is the higher figure, this will translate into approximately 12,000 gene sequences that we can both compare and anchor directly with the barley gene map and the rice and Brachypodium genomes to reach our objective. A considerable bioinformatics effort will be required to integrate and display the data and allow it to be queried.

Gene-based anchoring

In order to reduce the number of BACs to screen for each gene, we will use a series of complex multi-dimensional BAC pools that permit use of only a small number of assays to identify a single BAC clone.  These pools will be screened by PCR using primers specific to mapped genes. However we are investigating three other approaches. First, we will explore use of the Illumina Goldengate-based SNP mapping technology that we have used so successfully in AGOUEB to screen the BAC library with 1536 genes at a time. This process has already been demonstrated to be successful by Tim Close in UCR. Second, we will try an alternative and highly multiplexed screening approach using an Agilent microarray containing >44,000 barley unigenes to screen the BAC pools. Pilot studies aiming to anchor the wheat chromosome 3B physical map to the barley EST genetic map are very encouraging (Figure 1). Finally we will explore the use of next generation sequencing technologies for establishing the gene content of the BAC pools. We have no preliminary data on this approach (as of 1 August 2008).

Figure 1:  Barley 15K Agilent array probed with a reference mixed barley mRNA pool (RED) which allows grid alignment and spot identification and a single complex pool of c. 400 Wheat BAC clones from the chromosome 3B minimum tiling path (GREEN) (from C. Feuillet, INRA, Clermont-Ferrand). The green spots should identify homologous genes contained within the wheat BAC pool. 

Figure 2:  Searching the rice genome sequence for the orthologs of positive probes confirmed that between 45% and 70% corresponded to genes that are located on rice chromosome 1, which exhibits highly conserved synteny with wheat group 3 chromosomes. X-axis, A3 – D4 represent different BAC pools that were hybridised to the 15K barley array. Y-axis % positives whose orthologous sequence is located on rice chromosome 1 (Os01).