Developing a physical map of the Lolium perenne genome based on high-information content BAC fingerprinting and BAC-end sequencing

Grassland is a predominant crop in the UK and Lolium perenne (perennial ryegrass) is the single, most significant component of the majority of this grassland. In total, UK grasslands account for more than 50% of the land area and 70% of all agricultural land use and the market value of UK agricultural grassland in 2006, measured in terms of meat and milk production, is estimated to have been c. £4.8 billion. In addition to conventional agricultural use, grass and grasslands are of fundamental importance in amenity situations, including sports fields and landscaping, and there is also increasing interest in the development of alternative grassland uses, such as for biofuel and platform chemical production. IBERS is the major centre for ryegrass breeding/germplasm resources within the UK as well as being the focus of bioenergy grass improvement. It has an established infrastructure allowing the interplay of research in plant genetics, genomics, physiology, biochemistry and environmental analysis with breeding and sustainability objectives, thus, it is in a unique position to exploit new genome technologies. A major aim of grass scientists is to be able to define genomic regions that play an important role in determining target traits in these crops, as our ability to influence these traits (eg., drought/heat tolerance, nitrogen use efficiency, flowering, bioenergy traits) is fundamental to maintaining sustainable grasslands. A major step in comprehensive genome analysis is the establishment of a physical map. This involves a number of sequential processes: A) Develop extensive large-insert (BAC) genomic DNA libraries which, effectively, fragment and partition the genome into smaller pieces which can be catalogued. B) Generate distinctive 'fingerprints' of each of these BACs. C) Define the physical order of BACs within the genome by identifying overlapping fingerprint patterns (contiguous clones, or contigs). D) Relate the identified contigs to existing genetic or chromosome maps. Extra information can be obtained by sequencing the ends of each of the BACs. This information can be used in confirming overlapping fingerprint patterns and in helping to define the genome structure. The outcome is that the genome is broken down into an ordered series of manageable fragments. These can be used for targeted DNA sequencing of particular regions of the genome / or for large scale whole genome sequencing. The aim of this research is to develop this resource for grass and other monocot researchers to enable a greater understanding of the genome structure and function. In order to make this information widely available, an open-access web-interface will be developed which will display progress in the project and allow for the raw data to be downloaded, so that other researchers can use it in their own analyses.

IBERS is the major centre for ryegrass (Lolium spp.) breeding/germplasm resources within the UK. It has an established infrastructure allowing the interplay of plant genetics, genomics, physiology, biochemistry and environmental analysis with breeding and sustainability objectives. Thus, it is in a unique position to exploit new genome technologies in defining genomic regions with important roles in determining target traits. The principle aim of this proposal is to develop a physical map of the L. perenne genome using high information content fingerprinting (HICF) of a c. 10x L. perenne BAC library (241920 clones, estimated genome size 2 - 2.8 Gb) assembled using FingerPrint Contig (FPC) software alignments. Projecting from physical mapping projects in other plant species it is expected that this will produce 5000-10000 unordered contigs. Refinement of this physical map will be done by manual editing of the automated assembly and incorporation of BAC-end sequencing (BES) data from all 241920 clones. This process will identify: a) chimaeric contigs, b) conflicts between fingerprint and BES data and c) new contigs, by detailed analysis of fingerprint patterns of terminal clones of existing contigs. Manual editing may reduce the number of contigs by 50%. As part of this programme, BAC DNA pools will be developed from the 10x library suitable for PCR screening. Screening these pools with cross-species markers of known genetic and/or physical position in the Lolium and other monocot genomes, will align the physical contigs with existing genetic and cytological maps. Further comparative genomic information will be obtained using BLAST searches of the BES against other monocot resources, including a L. perenne GeneThresher (methylation filtered) sequence library. The developing physical map will be displayed using the WebAGCoL software via an open-access web-site. BES data will be submitted to EMBL/GenBank as well as being available via the project web-site.