Barley is an inbreeding diploid member of the Triticeae (that also includes wheat and rye) and has an established role as a model for genetic, and latterly genomic, research in monocotyledonous crops. We propose to integrate state of the art genomics technologies with advanced biological resources that have been systematically generated over the last 30 years, in a project (BARCODE) that represents a collision between historical mutation-genetics research and advanced genomics technology.

A total of 1533 k€ has been awarded by the Scottish Funding Council, DFG and MUR to:

• Robbie Waugh, SCRI, Dundee UK
• Nils Stein, Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany
• Michele Morgante, Dipartimento di Scienze Agrarie ed Ambientali, Universita' di Udine, Udine, Italy.

Within three clearly defined objectives of the BARCODE project we aim to:

1. Genetically map 480 morphological mutant loci onto the barley genome (at low to medium resolution). To achieve this we will combine use of a new high throughput gene-based SNP genotyping platform with an unique series of available nearly isogenic mutant lines.

2. Develop approximately 400Mbp of barley BAC-end sequence data. This will be linked directly to a High Information Content Fingerprint (HICF) BAC contig-based physical map generated in two parallel projects. Using the combined information from 1., 2. and the rice (maize and Brachypodium) genome sequence we will build physical BAC contigs in silico across a significant portion of the mutant loci.

3. Identify, isolate and validate at least three morphologically and / or developmentally important barley genes that are perturbed in specific mutant lines, demonstrating the utility of the linked genetic and physical map.

Progress at SCRI

Figure: Gene targets for the BARCODE project

Resources

The project will build upon ongoing research programmes in the partner organisations, benefiting from existing in–house and international informatics resources for storage, analysis and graphical display of genotypic and sequence data.

One especially notable collection of mutants is the ‘Bowman nearly isogenic lines'(Bowman NILs) developed by Professor Jerry Franckowiak over the last 20 years. The Bowman NILs are a collection of approaching 1000 morphological mutants that have been generated in a wide range of genetic backgrounds and then repeatedly backcrossed to cv. Bowman (the recurrent parent) to generate NILs. Professor Franckowiak's Bowman NILs contain meiotically purified mutant loci responsible for clear morphological and developmental phenotypes in a single common wild-type genetic background.

Over the last three years, we (SCRI, IPK and collaborators at UC Riverside in the US) have developed a high throughput SNP genotyping platform for barley based on the Illumina GoldenGate and Oligo Pool Assays (OPA).

Expected outputs of the project

Objective 1

The genetic location of up to 480 barley morphological and developmental mutant loci, referenced to the broader barley gene map and their comparative locations on the rice (eventually Maize and Brachypodium) genome sequence. This will provide regional gene content and candidate gene information. Our thesis is that this will represent a platform for gene isolation in barley, and stimulate hypothesis driven research in the cereal and forage grasses.

Objective 2

To generate the structural genomics information (BAC-end sequences) and resources required to develop physical contigs across the genetically mapped mutant loci. In the process we will make a major contribution towards the ‘barley genomics vision'. Despite being able to narrow down the location of each mutant locus to a relatively small genetic interval, identifying the lesion conferring the mutant phenotype will in most cases require adoption of a forward genetics strategy. Objective 2 involves the generation of BES information to facilitate the development of genetically anchored physical contigs spanning the mutant loci, and to make map based gene isolation in barley facile. Importantly, instead of focussing on each of the 480 mutant loci individually, which would require an enormous quantity of BAC library screening with linked gene.

Objective 3

To identify and validate at least three genes perturbed in specifically chosen mutant lines. Based on the size and location of mutant containing segments, we will attempt to isolate at least one gene responsible for a morphological phenotype, per partner, by forward genetics. Our choice of target genes will reflect the biological interests of the partners - grain development, morphology and plant architecture, but we will not realistically be able to make a sensible target selection before the BarleyOPA1 and 2 mapping information becomes available. We will however prepare for positional cloning by generating F1 hybrids between four to five of our top priority mutants (in each group) and the cultivars Bowman and Morex (before the start of the project). These will include gigas (large grain) glo-a (round grain), adp1-a (awned palea), bra-c (third outer glume), trp (triple awned Lemma), dsp (dense spike), zeo (zeocriton – dense ears), fst (fragile stem), mul2 (supernumery florets), com2 (branching inflorescence), cul1/2/3/4 (uniculm) and mnd (many noded dwarf) amongst others. Once the genotypic data becomes available, we will prioritise these targets based on genetic location, the putative size of introgressed segments, the amount of information available through comparative genetic analyses, the quality of the BAC contig information spanning the locus, the availability of additional independent mutants with the same phenotype (for example, from other Bowman NILs and the TILLING populations available in both IPK and SCRI).To achieve the required genetic resolution for positional cloning, large F2 populations (>2000 individuals) for target genes will be developed in each lab using the relevant F1's developed above.

Participants involved

• Large-scale crossing of the Bowman isoline: David Harrap, KWS UK Ltd
• Barley mutant stocks: Jerry Frankowiak, Warwick, Australia; Udda Lundqvist, Nordic Genetic Resources Center, Alnarp, Sweden

Presentations

• International Atomic Energy Agency symposium ‘Induced mutations in plants' Vienna, Austria August 2008, Arnis Druka
• Small Grains Cereals Workshop, Harpenden, UK April 2008, Arnis Druka
• Plant and Animal Genome Conference, SanDiego, USA January 2008, Robbie Waugh
• Barley Genome Net meeting, Salsomaggiore, Italy December 2007, Arnis Druka

Publications

Two new barley ERA-PG projects funded! The newsletter of UK Small Grain Cereals Research.