UK HEALTH & WEALTH FROM Brassica: DEVELOPMENT OF FUNCTIONAL GENOMICS RESOURCES, UNDERPINNING THE UK-Brassica RESEARCH COMMUNITY

A consortium has been assembled for the long term exploitation of Brassica in the UK. Following discussions amongst the UK Brassica Research Community (UK-BRC) it was decided that a contiguous physical map of the A and C genomes (representing UK Brassica oil and vegetable crops) is the essential first step in provision of resources and services to accelerate identification of gene function. This will provide the focus for future collaborative development and exploitation of results from the related crucifer Arabidopsis. The programme of work consists of construction of BAC contigs anchored to the Brassica genetic maps and the Arabidopsis sequence, with many anchors selected by members of the UK-BRC. A strong component of the programme is focused on promoting interaction and sharing of resources, data and expertise amongst the UK-BRC.

Proposal submitted to the BBSRC by the following principal investigators:

King/Beynon	Horticulture Research International
Bancroft/Trick	John Innes Centre
Kearsey/Jones	University of Birmingham
Scott	University of Bath

This forms the basis of the current BBSRC Investigating Gene Function Initiative project
(Proposal Co-ordinated and submitted by Dr Graham King, HRI, 13^th May 1999)
N.B. this document includes the general sections of the proposal - for clarity some detailed sections have been omitted.
_____________________________________________________________________________________________________________

18. DESCRIPTION OF THE WORK

Objectives of proposed work

Construction of contiguous physical maps for the Brassica A and C genomes

Integration between Brassica genetic and physical maps, functional loci and Arabidopsis genome

Early identification of genomic Brassica BAC contigs associated with gene families of direct utility to UK-BRC

Promoting interaction and sharing of expertise and resources amongst the UK-BRC

We are constructing a Consortium for the long term exploitation of Brassica in the UK. This proposal represents the first stage of a continuing dialogue amongst the UK Brassica Research Community (UK-BRC) and other stake-holders in the Brassica public and private sectors. The UK-BRC has decided that a contiguous physical map of the A and C genomes (representing UK Brassica crops) is the essential first step in provision of resources and services to accelerate identification of gene function. This will also provide the focus for future collaborative development and definition of a coherent UK research strategy. The UK-BRC brings together researchers from institutes, universities and the commercial sector and thus bridges the gap between fundamental research and practical applications. This proposal will underpin a positive growth in the UK-BRC. Brassica is particularly well suited to biotechnological approaches as demonstrated by the existing number of patents and investment worldwide.

The crucifer genus Brassica is unique in representing crops of importance to both arable and horticultural sectors of UK agriculture. Their reproductive biology and breeding systems allow ready manipulation of considerable natural genetic variation. The particular biological properties of brassicas have led to their wide adoption as a research material over many decades in the UK and elsewhere, together with associated development of key experimental resources. The close evolutionary relationship with the model species Arabidopsis thaliana provides a compelling argument for adoption of Brassica as the paradigm for transfer and testing of fundamental knowledge to crop plants. As a crop model Brassica species offer many unique targets for identification of gene function, including those associated with new product development and improved human diet. The complex genome organisation, typical of crop plants, is readily accessible, and will provide the insights required for understanding and manipulating higher order interactions at the level of the transcriptome, proteome and metabolome.

Background and introduction

Brassica species in the UK economy

Oilseed rape is grown on about 500,000 hectares in the UK, with the production of 1.5 million tonnes valued at £408m (MAFF, 1997). The value of the UK horticultural brassica crop is £184m (745,000 tonnes) from 39,000 hectares (MAFF, 1997).

Oil from rapeseed is the basis of industrial applications for margarines and other edible products for the processing industry (eg Unilever) and polymer processing for non-edible products. Horticultural brassicas are processed as frozen vegetable products, cauliflowers for pickles, baby-leaf brassicas for salad packs.

In the UK, relatively recently introduced brassica crops such as calabrese have seen considerable growth in the last 20 years, and have maintained the market share of brassica as a staple vegetable. UK consumers are likely to respond well to the availability of fresh products with increased health benefits. UK vegetable production is under increasing pressure as a result of buying patterns and continuous supply requirements of multiple retailers; many of these issues can be addressed by genetic improvement.

The scientific value of Brassica

Brassica species possess attributes which in combination provide a rich source of unique opportunities for scientific study and economic development. These include:
The primary temperate oilseed crop, widely used in the processing industry, with current developments for alternative fuel oils, high-grade polymers, vaccines and other products.

high homology with the Arabidopsis complete genome sequence

Health properties: epidemiological studies have emphasised the beneficial role of Brassica in diet. Health benefits which include reduction of coronary heart disease and cancers are likely to arise from glucosinolates, folates, phenolics, with possible contribution from other secondary metabolites.

Glucosinolate synthetic pathways leading to accumulation of a series of distinct products with properties useful for: anti-carcinogenic diet, bioremediation of soils, feedstocks in chemical industry, and molecular 'pharming'.

Ready availability of large quantities of specific plant tissues and organs is of relevance to many fundamental research studies.

Extensive genetic diversity, including a range of domesticated natural mutants resulting in the distinct morphological forms used in horticulture (cabbages, cauliflower, broccoli including Calabrese, Brussels sprout, Kohlrabi, kales, Chinese cabbages etc). A wide range of germplasm also exists in B.napus, with diversity extended through development of re-synthesised lines where production of amphi-diploids provides access to the diversity of diploid relatives.

UK investment into collection and maintenance of Brassica germplasm at the HRI Genetic Resources Unit has led to establishment of a world-leading collection. This proposal underpins efforts for further utilisation of the collection and associated information.

Well characterised inter-specific relationships based on 'triangle of U', with methylation status and relationship to Arabidopsis indicate that it is well-suited to studies of genomic imprinting.

Amenable to high throughput microspore culture, transformation and tissue culture

the model sporophytic self-incompatibility system

There is already considerable synergy between Brassica and Arabidopsis research. As well as the complete genome sequence and mutant resources informing research in Brassica, we expect Brassica to enhance the Arabidopsis programme by providing:

functional targets for gene identification

relative ease of biochemistry or proteomics, metabolomics and transcriptome analysis.

clues to search for phenotype in Arabidopsis. Providing specific targets for Arabidopsis research (e.g. oil biosynthetic genes)

1. Competitive and strategic situation, and potential for growth

The UK has established a world-leading reputation for genetic and genome analysis of brassicas, with development of high quality resources and expertise. In the last few years there has been increasing research activity in this field overseas, with particularly large investments in Canada, France and Asia. It is important that the UK research community is able to compete internationally through development of distinct high quality resources and data sets.

We intend to establish Brassica as the paradigm for transfer of fundamental knowledge from Arabidopsis to crop species via functional genomics. The resources and services developed in this proposal will be available for future exploitation by BBSRC, MAFF and industry and form the focus for future proposals from the UK-BRC. We expect the commercial and social importance of brassicas in the UK to be extended, especially through the development of new products and increased awareness of health benefits.

Benefits from functional genomic studies in both Brassica and Arabidopsis may be readily exploited in Brassica. Brassica has proven to be well suited to genetic improvement either through conventional Marker Assisted Selection or Genetic Modification.

Marker Assisted Selection

The development of a physical map will underpin characterisation of gene function leading to efficient marker assisted selection. Previous work has included characterisation of the multi-allelic self-incompatibility system and development of broccoli with enhanced nutritional value. As examples, in the latter case the UK benefited from licensing of intellectual property to the USA, in the former case via income from technology transfer to UK and multi-national companies.

Genetic Modifiation

This proposal will also underpin development and understanding of genetically modified crops. The strategic relevance of Brassica as a crop model lies in the properties of a partially methylated genome with replicated loci possessing gene sequences of high homology to Arabidopsis. Research providing greater insight into transgene stability and position effects will be possible once we are in the possession of a contiguous physical map for Brassica. In combination with the UK expertise in high quality, marker-free transformation systems, and improved understanding of reproductive systems, this should enable a more informed dialogue with regard to risk assessment.

Health attributes of Brassica vegetables

The positive health effects of eating fruit and vegetables are now widely recognised. Epidemiological studies (eg Block, 1992) have provided impressive evidence that consumption of fruit and vegetables reduces the risk of coronary heart disease and cancer, and ensures sufficient intake of minerals and vitamins to maintain health. Brassica crops comprise a highly significant part of vegetable intake in the UK. The mechanisms by which vegetables in general, and Brassica in particular have these attributes is not fully understood. However, it is likely to be due secondary metabolites, such as flavanoids and other phenolic compounds, acting as antioxidants, and glucosinolates and other sulphur containing metabolites such as S-methyl cysteine sulfoxide, acting as anticancer agents due to their ability to induce detoxification enzymes in mammalian cells and to reduce the rate of tumour development. Studies on glucosinolates have demonstrated (1) the extensive genetic variation which occurs in Brassica germplasm collections and (2) the use of Arabidopsis as a model system to elucidate genetic regulation and facilitate gene cloning. Thus, it has been possible to develop broccoli with enhanced anticarcinogenic activity through marker-assisted breeding programmes. This approach could be adopted for many other metabolites in Brassica with associated health benefits to the UK consumer, and opportunities for development of novel products by UK commerce. However, for these programme to be successful, we need access to the tools which will be developd in the proposed programme. (Block G (1992) Nutrition and Cancer 18, 1-29)

Further opportunities arise from the fact that synthesise a wide range of the secondary sulphur metabolites, glucosinolates by Brassica species, have additional desirable properties in agricultural production (e.g. through bio-remediation of contaminated soils, etc). Additional potential exists for a wide range of neutraceutical and functional food applications in both the vegetable and oilseed sectors.

Wealth production from Brassica

The ready production and properties of Brassica oils has led to considerable investment world-wide in the improvement of existing lipid profiles and development of new oil-based products - both edible and non-edible. ACTIN, the Alternative Crops Technology Interaction Network is a collaboration between UK Industry and Government to stimulate novel technologies to meet industry demand for alternative, i.e. non-food crop products, thus enhancing wealth creation in the UK.

As an example, adipic acid is used for the manufacture of a wide range of polymers including high-grade engineering plastics. It has a global market in excess of 2.5 million tonnes worth over £1 billion. At present, adipic acid is derived from mineral oil by a process which releases gases, such as N₂O that damage the ozone layer and contribute to global warming. If the UK only produces 5% of the global requirement for adipic acid from oilseed rape this would require about 500,000 hectares of arable land, an area similar to that of the current oilseed rape crop.

Research described in the Appendix ("UK-BRC directory") highlights the potential for growth in this area based on a range of biosynthetic pathways and metabolites, and emphasises the key role Brassica has to play in exploitation of functional genomic approaches.

Maximising the potential from UK investment in Arabidopsis

Over the past decade, the UK has developed and maintained a reputation for high quality plant science which has focused on the model dicot Arabidopsis. This has been encouraged through a series of BBSRC initiatives and involvement in international sequencing projects. The NASC is an international centre for management of Arabidopsis material and data curation. We will make direct use of shared resources with the Arabidopsis programme proposed under this IGF initiative.

UK-BRC will be able to exploit the vast range of fundamental information from Arabidopsis directly through alignment of the complete genome sequence with Brassica physical maps. Thus the construction of Brassica contiguous physical maps becomes the fundamental resource for development of crop-based functional genomics. The rate at which advances have been made world-wide, and the current level of investment, indicate the urgency with which the UK must develop and exploit this resource.

Many research groups currently working with Brassica are actively involved in the Arabidopsis community. We see this relationship continuing to flourish. The Brassica functional genomics programme will build on the existing close relationship with the Arabidopsis programme in order to develop a more interactive flow of information. In particular we expect an increasing trend with feedback of results and hypothesis testing between Brassica and Arabidopsis. As an initial example we would be able to test the efficacy of Arabidopsis micro-array and expression chips to detect and characterise gene expression in Brassica.

As these technologies become more cost-effective we would expect to test the converse situation. In particular there is a reasonable expectation that an organism with more complex genome organisation, such as Brassica, displays a greater range of developmental responses via modulation of gene expression. We thus expect detailed comparative analysis with Arabidopsis will allow a more subtle appreciation of the complexities of gene regulation at the level of the transcriptome.

The UK Brassica Research Community (UK-BRC)

Distinctness of the UK-BRC

We stress that this commonality of resources will become a distinguishing feature and benefit for the UK plant science community as a whole. Whilst the US public and private sector functional genomics programmes for Arabidopsis are well advanced and well funded, they are not well integrated with the agricultural programmes, with a particularly low level of investment in Brassica. A concerted effort and large investment in Brassica genomics research has been initiated in Canada, but this at present is less well integrated with the Arabidopsis community. In Europe, Rhone-Poulenc and Limagrain have constructed a consortium with the French public sector research organisations which is poised to benefit from integration of Arabidopsis and Brassica research.

It is important that the UK can maintain a distinctive and unique position in this rapidly developing area. Amongst the UK-BRC we have been successful in attracting investment from a range of UK and multinational industrial sponsors. Whilst the UK is currently competitive in research for both of these crucifers, this position will be lost unless we capitalise on the potential for growth, which is immense and imminent. There is also scope to exploit unique properties of other UK crucifers such as Isatis (woad, for indigo dye production).

Development of the UK-BRC

BBSRC has over the years invested in establishment of particular research strengths focused around Brassica species. Research networks and centres of excellence have been built around expertise in characterisation of the sporophytic self-incompatibility system, parasite resistance, detailed genetic mapping and genome analysis, biometrical genetics and quantitative trait analysis, lipid biochemistry, glucosinolate synthesis, physiology, and morphological development including the various horticultural forms and pod-shatter. Much of this work has been facilitated by the excellent diversity accumulated in UK genetic resource collections, of which the vegetable gene bank at HRI is an international repository. This is complemented by the UK leading efforts to establish high quality populations for genetic mapping and gene identification. Efficient and marker-free transformation systems have been developed for the major crop types and experimental materials.

Existing Resources

The UK-BRC currently consists of scientists funded by BBSRC, NERC, MAFF, industry and levy bodies. Existing resources, expertise and activities relating to genetics, genomics and gene function for Brassica in the UK include:

Unparalleled series of reference doubled haploid mapping populations for Brassica species, together with associated integrated linkage maps, marker probes and large insert BAC libraries.

Series of related substitution lines for B.oleracea genome

Large number of well characterised single and quantitative trait loci

Molecular genetic characterisation of a wide range of gene families associated with a range of functions

Extensive genetic resources and associated data

Doubled haploid self-compatible rapid cycling lines readily amenable to microspore culture and transformation.

Efficient transformation systems

A SSR development programme

Pathogen collections associated with the major diseases

Databases associated with the above resources

By leading the effort to develop the Resources and establish Services, the institutes will facilitate the process of university research groups being able to exploit their expertise and extend their fundamental studies from Arabidopsis to Brassica. The institutes are well placed to provide a service to enable this to happen. Examples of this are highlighted in the "UK-BRC directory".

Different subsectors of the UK-BRC have worked together over the past two decades in a number of collaborative ventures. There has been a long association between the University of Birmingham and Horticulture Research International in genetic analysis, and between UoB and JIC. HRI and JIC have collaborated with a range of university departments throughout the UK (including UoB, Warwick, Nottingham, UEA, London, Wye, Oxford, Durham, Reading). Individual universities and institutes are involved in a wide range of research contracts with different sectors of the UK Brassica industry.

These ongoing relationships are now becoming more formalised in the UK-BRC. We have established a UK-BRC electronic Mailing List in order to share information, both that associated with provision of resources and services for functional genomics, and also as a forum for dissemination and discussion of information amongst the multi-disciplinary Brassica community.

Breadth of the UK-BRC

We have started to identify the key activities and requirements of the UK research community through compilation of the provisional "UK-BRC Directory" (Appendix). This document presents responses from a recent survey of UK Brassica researchers, and demonstrates the scope and depth of expertise within the UK. It also underlines the key position of functional genomics resources and services in maintaining a competitive position for UK Brassica researchers.

We expect the membership to diversify as fundamental findings from Arabidopsis find their first crop applications in brassicas. Research laboratories currently working with Arabidopsis will find they are able to make ready use of their existing resources, gene probes and screening techniques on Brassica experimental material. The trait and gene-based research described in the "UK-BRC directory" is complemented by a wider community of agronomists, plant physiologists, pathologists, entomologists and other scientists.

4. Defining the priorities

An initial meeting was held at HRI between representatives from HRI, JIC, Birmingham, Bath, Nottingham, Biogemma, with consultation of others not able to attend. At this and a subsequent meeting it was decided that development of a physical map was an essential first step in the development of the UK-BRC resources. At that time it was perceived that the next likely priority would be development of Brassica EST resources. The information compiled in the provisional "UK-BRC directory" substantiates this view. We envisage being able to move towards this secondary goal in the next two years. The subsequent decision making process will now be formalised through establishment of a UK-BRC Steering Committee, providing a focus for community discussion and involvement. Subsequent collaborative developments may include testing the efficacy of Arabidopsis micro-array and expression chips to detect and characterise gene expression in Brassica; wide availability of efficient micro-spore transformation; development of transcriptional transactivation resources.

5. Establishing a Steering Committee

Through discussions amongst the UK-BRC, using the mailing list as a focus, a provisional UK-BRC Steering Committee for functional genomics resources is being established to set priorities for use and deployment of the resources and services. We will also establish further regular meetings of the UK-BRC to encourage dialogue and discussion of requirements, priorities and opportunities. The remit will include setting priorities in order to shape the community programme. Its activities will thus not be confined to this proposal.

Business Plan for development of resources and research networks

In the short-term the resources and services developed in this proposal will be available immediately throughout the community. We are involving the UK-BRC from the earliest stages by inviting proposals for nominating probes of interest to be anchored to the BAC contigs. Once the contigous physical maps have been generated for Brassica, we expect there to be an increasing requirement for informatics development associated with the Arabidopsis genome sequence and functional genomics resources. We expect the initial work to stimulate a range of further proposals which will be developed amongst the UK-BRC.

The Steering Committee will play an important role in determining the realistic scope for development of resources and to investigate gene function in Brassica.

Involvement of the UK-BRC, and benefits to the UK-BRC in this proposal

All resources developed will be free of Intellectual Property costs to the public sector researchers in the UK. Resources will be made to others available by licensing on a non-exclusive basis. Outside the UK resources will be provided at F.E.C. We plan to distribute resources (BACs, filters, etc) from JIC. However in the longer term this function could be taken over by NASC.

In the long term (5-10 years) we foresee an increased use and dependancy upon common resources both in the UK and world-wide. A cycle of re-investment can be set up through licensing agreements with the commercial sector. The development of a concerted effort amongst the UK-BRC will be used to attract wider international investment.

6. PROJECT PLAN

6.1. Rationale

The group of crops most closely related to the key model plant Arabidopsis thaliana (Arabidopsis) are the brassicas. They are therefore the obvious choice as the first group of crops for which methods need to be established by which Arabidopsis genomics, its genome sequence, and all aspect of functional genomics can best be applied to crops. It is widely held that Brassica functional genomics should be developed based initially upon the aim of maximising our ability to utilise Arabidopsis functional genomics.

6.2. State of the art

To date, Brassica genomics has focused on the development of genetic maps. Investigations of the structure of the genomes of Brassica species, in relation to that of Arabidopsis, indicate that the genomes of diploid brassicas approximate to a triplicated and extensively rearranged Arabidopsis-like genome (Lagercrantz, 1998 Genetics 150:1217-1228). The genome of the highest value Brassica crop in the UK, oilseed rape (Brassica napus), which is an amphidiploid (AC), is known to be composed of the genomes of the diploid species B. oleracea (C) and B. rapa (A), so approximates to six equivalents of an Arabidopsis-like genome. These previous analyses could use only genetic mapping approaches in the brassicas, so analysis of genome structure is both superficial and, due to lack of polymorphism at some loci for most molecular markers, incomplete. Therefore, until very recently, we had no appreciation of the extent of conservation of genome structure on a gene-by-gene scale (microsynteny) between either the paralagous regions within the Brassica genome or between those and their homoeologous regions in the Arabidopsis genome. The previous assumption had been that, as synteny could be detected over large genetic segments (tens of cM), microsynteny would be almost perfect.

6.3. Recent findings

Using new molecular tools, in particular two BAC libraries (using different vectors and with different average clone insert sizes) made from genomic DNA of Brassica oleracea A12DH, we have begun to investigate detailed genome structure in defined chromosome segments homoeologous to sequenced regions of the Arabidopsis genome. Results from several of our labs indicate extensive loss of microsynteny between the paralogous segments of Brassica chromosomes, and between those and their Arabidopsis homoeologs (see Fig 1). There is also indication of differential expansion of some paralogues relative to others and their Arabidopsis homoeologues. This variance from the expected high degree of conservation of microsynteny with Arabidopsis makes imperative the immediate development of modern Brassica genomics resources within the UK (as are presently planned in both Canada and France), in order to maintain a credible competitive presence of UK academic and industrial scientists in the area of Brassica-based science and Brassica crop improvement.

6.4. The need to generate resources to underpin the efficient assignment of functional information to genes in Brassica species.

The UK Brassica Research Community (UK-BRC) has identified as the most urgent requirement for functional genomics in brassicas, the construction of physical maps, based upon BAC clone contigs, representing the genomes of B. oleracea and B. rapa (thus, in combination, B. napus). These physical maps will be anchored in detail to both the Brassica genetic maps and the Arabidopsis genome sequence. The underlying BAC clone libraries will represent a resource from which genomic clones Brassica genes can easily be obtained. The maps will represent a conduit for both the application of Arabidopsis functional genomics in brassicas, and for the utilisation of brassicas for the efficient development of many aspects of functional genomics in Arabidopsis

6.5. Proposed Programme of Work

Following discussion amongst the UK-BRC, as a first step we propose that 4 research institutions are involved in generating the resources to underpin the efficient assignment of functional information to genes in the brassicas. In addition to serving the whole UK-BRC by establishing the underpinning resources that the community requires, we will structure the programme so as to provide more immediate services and support to individual laboratories:

BAC clones and all data will be accessible to the community throughout the project.

Involvement of community from the first stages in the project, through selection and proposal of key gene loci from Arabidopsis and Brassica to be used in BAC characterisation.

Two of the participants in the project, HRI Wellesbourne and JIC, have established resources and expertise in plant genomics. They will conduct a large proportion of the work (for cost-efficiency and speed), but will also have major training and resource distribution roles. An important element of the project is to ensure that the UK-BRC capitalise on the resources which have accumulated from previous investment in institutes, by facilitating the flow of expertise and materials throughout the community.

In the following sections we outline our objectives, strategies, cost calculations and describe our long-term plans for provision of materials.

6.6. OBJECTIVES

Construction of contiguous physical maps for the Brassica A and C genomes

Integration between Brassica genetic and physical maps, functional loci and Arabidopsis genome

Early identification of genomic Brassica BAC contigs associated with gene families of direct utility to UK-BRC

Promoting interaction and sharing of expertise and resources amongst the UK-BRC

6.7 Resources

To date, two Brassica BAC libraries have been constructed and are available to UK scientists. Both were constructed using Institute CSG funding, and both were constructed from genomic DNA of the doubled haploid B.oleracea A12DH (var. alboglabra). These libraries are: 26,112 clones x 120 kb average insert size in the vector pBeloBAC11 (BoB, constructed at the Texas A & M BAC Center by CDR from HRI-Wellesbourne) and 33,792 clones x 145 kb average insert size in the transformation-competent vector BIBAC2 (JBO, constructed by the group of I.B. at JIC). Before the proposed research programme commences, JIC will produce a library of genomic DNA of the doubled haploid B.rapa RO18 (JBR) to consist of 36,864 clones x 145 kb average insert size. All three libraries will be utilised in the programme.

The finished physical maps will be based upon fingerprint-assembled BAC contigs. The fingerprint contigs will be assembled automatically only, using assembly parameters set to high confidence limits. The highly labour intensive manual editing required to construct larger contigs will be eliminated since we are making direct use of the Arabidopsis sequence resource. The C-genome BoB and JBO libraries will both be fingerprinted and contigs assembled in a single "project". The A-genome JBR library will be fingerprinted and assembled as a separate "project". We estimate that average contig sizes will be approximately 450 kb.

The fingerprint-generated contigs will be oriented and ordered relative to each other by anchoring to the Arabidopsis genome sequence by hybridisation of probes specific to Arabidopsis genes identified in the genome sequence to high density colony filters of the JBO and JBR libraries. This will be conducted at 1333 positions in the Arabidopsis sequence, which will be equivalent to anchoring at 4000 positions in the Brassica genome (assuming an average genome triplication). Thus the anchoring will be performed at an average density of three anchor points per contig (assuming a genome size of 600 Mb for diploid brassicas). We consider this density to be the minimum necessary to allow us to deal with the extent of divergence of microsynteny that we have observed. The contigs will also be anchored to the Brassica genetic maps by hybridisation screening of the BAC libraries with at least 200 sequenced Brassica RFLP markers.

Results from an existing project (BBSRC Grant ref. 6/G11431), which aims to integrate the genetic and cytological maps of B.oleracea via chromosomal-Fluorescent In Situ Hybridisation (FISH) analysis, will be used to validate and position BAC contig assignments in terms of distribution on the karyotype. As both Birmingham University and HRI are involved in this project, we will ensure that the current proposal benefits from an integrated information flow, especially in the early stages of identifying unambiguous anchor probes.

7. Service to the UK Brassica Research Community

The consortium formed to conduct the proposed experimental work includes scientists from four key UK institutions involved in Brassica research. The research is to be conducted for the benefit of the whole UK-BRC, as outlined in the "UK-BRC directory". All data will be made available by the Informatics group, using the WWW, as rapidly as feasible, with monthly releases of collated probing results giving BAC co-ordinates. Clones from the Brassica BAC libraries will be freely available for academic research during and after the project (as they are now) and will be made available for industrial use by non-exclusive licensing. A specific service to be offered to the UK-BRC will be through allowing them to contribute towards the selection of genes for anchoring of the Brassica BAC contigs to the Arabidopsis genome sequence. This could be exploited by members of the research community to, for example, identify genomic clones of Brassica genes (which will be in transformation-competent vectors) or for the construction of physical maps of defined regions of the Brassica genome that containing a gene specifying a trait of biological or commercial interest. Although generated to support specific projects, these data would be included in the systematic data set, thus maximising cost-efficiency by eliminating redundant analyses. Contig information from the fingerprinting exercise should appear at the end of the first year. Information describing significant contigs (of >4 BACs) will be released, allowing an emerging view and utilisation of work to date in physical map construction.

9. Deliverables

Long-term access to data and resources

The availability of the contiguous physical map is the first stage in the development of a full suite of integrated functional genomics resources and tools for Brassica. We expect to attract investment for a concerted development of high quality EST sequences over the next two years, which will be integrated with a bid for funding. This will utilise the expertise of the wider UK-BRC and integrate directly with the physical maps being constrcuted in this programme.

We consider efficient long-term access to the map data and BAC libraries to be very important issues. The data will be archived both within BrassicaDB, which we expect will become the primary database for the UK-BRC. They will also be copied to databases at HRI Wellesbourne and in the USA, and will be made available to any Brassica (or plant) database superseding BrassicaDB.

By providing long term, high quality access, the clone libraries and physical map data based upon them will continue to provide added value to UK plant functional genomics projects and will become a permanent, vital and very efficiently accessed resource supporting plant science world-wide.

Integration with existing and future work

Several projects in the current GAIT programme are investigating the transfer of gene organisation and associated functional information from Arabidopsis to Brassica. The current approaches utilise existing unordered BAC and other mapping resources. However, progress is slow for individual gene loci. We expect these studies to become routine through the development of the contiuguous physical map, and thus accessible by a greater number of Brassica and other researchers. The demand for detailed locus specific information will increase as the facility of carrying out high-throughput transcriptome and metabolome analysis in Arabidopsis increases. The information gathered in the "UK-BRC directory" provides a very clear indication of the extent to which development of these resources will benefit the UK-BRC in the future.

We will be widening the access to advanced technologies, particularly ensuring rapid flow of results and expertise between the Arabidopsis and crop communities. As mentioned in the introduction, Brassica is unique in utilising a large range of the natural genetic diversity within different crop sectors.

Summary

The construction of these resources, and the facilitation of effective public access, will add greatly to the value of the Arabidopsis functional genomics project and will help establish a broad, co-ordinated strategy for the development of plant functional genomics in the UK. We expect this initiative to act as a catalyst for economic development. The particular properties and products which distinguish Brassica species ensure that it is an excellent paradigm for crop plants in the functional genomics agenda.

The BBSRC and the UK government need to ensure that they maximise the value from any investment in functional genomics. We have outlined the scientific and economic arguments which illustrate the unique position of Brassica and UK-BRC in being able to capitalise on the continuing investment in Arabidopsis. We propose a model for the UK whereby the Arabidopsis research community and UK-BRC become increasingly integrated and benefit from re-investment arising from applications in Brassica.