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General enquiries on this form should be made to: Defra, Science Directorate, Management Support and Finance Team, Telephone No. 020 7238 1612 E-mail: [email protected] SID 5 Research Project Final Report Note In line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects. 1. Defra Project code 2. Project title This form is in Word format and the boxes may be expanded or reduced, as appropriate. 3. ACCESS TO INFORMATION The information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors. SID 5 (Rev. 3/06) Project identification NF0532 Brassica alboglabra -developing a new oil crop for the UK Contractor organisation(s) Warwick HRI University of Warwick Wellesbourne Warwick CV35 9EF 54. Total Defra project costs (agreed fixed price) 5. Project: Page 1 of 11 £ 44,893 start date ................ 01 January 2007 end date ................. 31 March 2007 6. It is Defra’s intention to publish this form. Please confirm your agreement to do so. ................................................................................... YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow. Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer. In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. (b) If you have answered NO, please explain why the Final report should not be released into public domain Executive Summary 7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work. The work carried out in this three month project had the aim to demonstrate the feasibility of developing Brassica alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for the UK. The project had three objectives: 1. To demonstrate the existence of natural variation within B.oleracea and related wild species for seed oil fatty acid profile and seed oil content. 2. To identify allelic variation for genes associated with fatty acid biosynthesis. 3. To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying the gaps in knowledge required to produce that ideotype. The results described below clearly indicate that it would be feasible to develop a Brassica oleracea oil crop for the UK. We have demonstrated the existence within the C genome genepool (B. oleracea and related wild species) greater natural variation for seed oil fatty acid profile and seed oil content than was found in the Ac (B. napus) genepool. We have also demonstrated allelic variation for genes known to be associated with fatty acid biosynthesis in the C genome genepool. This provides the underpinning basis for developing an oil crop in which cultivars are bred to produce oils tailored for specific industrial end uses. The ‘plasticity’ of the C genome as demonstrated by the many and varied crop morphologies within the species and also the variation in morphology between the wild relatives provides the genetic basis for redesigning a crop ideotype to provide a new oil crop which meets stakeholder needs in terms of agronomy, yield and reduced (potentially beneficial) environmental impact. However, a more extensive follow up project would be required to underpin. This would exploit the range of experimental genetic and genomic resources in C genome brassica available at Warwick HRI in a pre-breeding project to deliver plant lines with combinations of alleles to give optimised fatty acid profiles for particular end uses and optimised sustainable crop production i.e reduced and potentially beneficial environmental impact. These could be subsequently exploited in a programme to breed a novel (B oleracea) oil crop for the UK or delivered to the current UK oil seed breeding community by introgressing them via interspecific crosses and embryo rescue into a common ‘crop portal’ which would be an experimental line(s) derived from a well adapted cultivar(s) to be identified by the OSR group of the British Society of Plant Breeders. SID 5 (Rev. 3/06) Page 2 of 11 Objective 1: To demonstrate the existence of natural variation within B. oleracea and related wild species for seed oil fatty acid profile and seed oil content. Natural variation for seed oil fatty acid profile and content was demonstrated by analysing the seed oil of 10 seeds of each of the gene bank accessions used to make the diversity sets for cultivated forms of Brassica, oleracea and 14 related wild species (oleracea; alboglabra; atlantica, balearica; bourgaei; cretica; hilaronis; incana; insularis; macrocarpa; montana; robertiana; rupestris; villosa) which are being produced in HH3723 and the diversity set for B. napus which is being produce din the OREGIN project. Significant variation was found between accessions both for the range and amount of different fatty acids. The greatest observed variation was between the accessions in the wild C genome diversity set with the least variation being found in the B.napus set. This can be explained due to the effect of strong selection pressure which has been exerted by breeders on the modern B. napus cultivars for high oleic acid and low erucic acid to meet market needs; whereas the wild C genome species will not have been subjected to such strong unidirectional selection. The domesticated C genome genepool (B.oleracea diversity set) displayed less variation than the wild species suggesting that some variation may have been lost during domestication. Objective 2: To identify allelic variation for genes associated with fatty acid biosynthesis: A number of genes involved in the biosynthesis pathway of fatty acids have been identified in the model dicotyledon plant and member of the Brassicaceae, Arabidopsis thaliana. Three of these genes which are important in fatty acid synthesis: fatty acid elongase (FAE) and two fatty acid desaturases (FAD2 and FAD3) were sequenced in 188 accessions of the B. oleracea diversity set, and 96 accessions from both the wild C genome species and the B.napus diversity sets. The sequences were analysed for changes which may affect protein structure and hence enzyme function i.e. have an effect on fatty acid profile and content. A significant number of different alleles were identified for each gene. The wild C genome species DFFS and B oleracea DFFS sets showed a similar range of allelic variants with the B.napus DFFS set exhibiting the least diversity. Objective 3: To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying the gaps in knowledge required to produce that ideotype. There is a large diversity of crop morphology within B. oleracea; there is therefore potential to manipulate crop architecture in order to optimise the crop. In addition there are the tools and resources available in B. oleracea to exploit genetic variation for important input traits such as nitrogen, phosphorous and water use efficiency as well as pest and disease resistance. It is therefore possible to ‘design’ and breed to an ideotype to maximise the sustainability of the crop as a renewable feedstock. The current information relating to vegetable forms of B. oleracea was reviewed together with information from OSR studies during a ‘brain storming’ session to identify where it is applicable to the development of an oil form of the crop. This clearly demonstrated the potential of utilising the greater ‘plasticity’ of the C genome (as opposed to the napus genome) to breed a crop with many desirable traits. Although the C genome contains many gene duplications and triplications the effort required to understand and manipulate plant morphology will be easier in this ‘diploid’ species than in an allotetraploid such as B napus where the degree of gene duplication is doubled. Aspects of the crop which will were examined included: Spring sown v autumn sown crops (vernalisation requirement) Canopy structure/branching Flowering time Harvest index Height Root architecture Energy balance/efficiency (input v output) Yield Pod shatter Pest and disease resistance Resource use efficiency Breeding system (hybrid v inbred line) These were considered from a viewpoint of adaptation to climate change, carbon balance, economics, land use, environmental footprint and public perception. SID 5 (Rev. 3/06) Page 3 of 11 Project Report to Defra 8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer). The work carried out in this three month project had the aim to demonstrate the feasibility of developing Brassica alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for the UK. The project had three objectives: 4. To demonstrate the existence of natural variation within B.oleracea and related wild species for seed oil fatty acid profile and seed oil content. 5. To identify allelic variation for genes associated with fatty acid biosynthesis. 6. To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying the gaps in knowledge required to produce that ideotype. Objective 1: To demonstrate the existence of natural variation within B. oleracea and related wild species for seed oil fatty acid profile and seed oil content. Diversity fixed foundation sets (DFFS) are being developed under HH3723 for cultivated Brassica oleracea and 14 wild related wild ‘C genome’ species (oleracea; alboglabra; atlantica, balearica; bourgaei; cretica; hilaronis; incana; insularis; macrocarpa; montana; robertiana; rupestris; villosa). These are defined as “Informative sets of genetically fixed (true breeding) lines representing a structured sampling of diversity within the relevant genepool” (http://www.brassica.info/diversity/diversity_sets.ht). Each line within the DFFS is being derived from a single accession from the Genetic Resources Unit (GRU) international brassica collection at Warwick HRI and these ‘founder’ accessions were used as the experimental material for the survey of fatty acid profile in seed oil and oil content. Ten seeds of each accession were obtained either from the GRU (for B. oleracea accessions) or from S1 seed lots produced by self-polinating the C genome wild species founder plants in HH3723. All seed were equilibrated at 150C and 15% relative humidity prior to sampling. Individual seeds were weighed in 2 mL glass analysis vials with sealable lids. At the sampling stage seed were handled with nitryl gloves to reduce contamination. Lipid fatty acids were converted to their methyl esters (FAMEs) using the direct transmethylation method and GC equipment as described by (Larson and Graham, 2001), with some modifications. To minimize seed to seed variation, 10 seeds were analysed for each accession and processed through the transmethylation procedure. Heptadecanoic acid (100 µg) was added as an internal standard before processing. Cooled transmethylated samples were transferred to microfuge tubes and extracted three times with 200 µL hexane, with 1 min centrifugation at 14000xg between each extraction step to clarify the partitioned layers. The hexane fractions were pooled, dried in vacuo for 10 min and reconstituted in 1 mL fresh hexane. This procedure demonstrated comparable quantitative extraction for 1-5 seeds, with no losses for FAMEs with acyl chains >C10 (data not shown). A 2 µL aliquot was injected for FAME analysis. Fatty acids were identified and quantified by comparison to a 37 FAME mix (Supelco), using Chromquest 2.53 software (Thermo). The absolute and relative amounts of each fatty acid were calculated. SID 5 (Rev. 3/06) Page 4 of 11 The analysis of fatty acid profile demonstrated there was significant variation between accessions for the range and amount of different fatty acids. Figure 1 shows examples of the 22:1n 9 18:1n9 c 20:1n 9 18:3n 3 18:2n6 c B. oleracea gemmifera (Brussels Sprout) 18:1n9 c 18:1n9 c 22:1n 9 18:2n6 c 22:1n 9 18:3n 3 18:3n 3 B. oleracea italica (Inbred Broccoli) 18:2n6c B. oleracea italica (Doubled haploid Broccoli) Fig 1: % FA mol variation between Brassica oleracea accessions variation seen in fatty acid profile between accessions of cultivated B. oleracea and figure 2 shows the variation seen in the amount of an individual fatty acid (18:2n6 - Omega 6 ) as a percentage of the total seed fatty acid content. 35 30 Fig 2. Amount of 18:2n6 (Omega 6) as a percentage of the total seed fatty acid content in 192 accessions of B oleracea %mol 25 20 15 10 5 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 Plant line Figure 3 further demonstrates the natural variation found within three genepools: the wild C genome genepool (fig 3a) the cultivated C genome (B. oleracea) genepool (Fig 3b) and the cultivated AC (B. napus) genepool for an individual fatty acid; this time for Omega 3 (18:3n3) fatty acid. SID 5 (Rev. 3/06) Page 5 of 11 Fig 3. Amount of 18:3n3 (Omega 3) as a percentage of the total seed fatty acid content in a) Brassica spp “C” genome DFFS (Wild species) set 1 35.00 30.00 %mol 25.00 20.00 15.00 10.00 5.00 C 07 0 C 01 07 0 C 18 07 0 C 11 07 0 C 13 07 0 C 21 07 0 C 28 07 0 C 22 07 0 C 29 07 0 C 31 07 0 C 39 07 0 C 38 04 0 C 31 07 0 C 51 07 0 C 61 04 0 C 57 07 C 062 07 07 C 9A 07 0 C 59 07 1 C 21 07 0 C 55 07 0 C 63 07 0 C 56 07 0 C 89 04 0 C 90 04 0 C 93 04 0 C 87 04 08 4 0.00 b) Brassica oleracea DFFS set 1 30.00 %mol 18:3n3 25.00 20.00 15.00 10.00 5.00 ge ra m m go ifer a ng ylo de s ita lic a ita lic a ita lic a ita lic a ol ife ra tro nc hu da ta ge m m ife ta pi ta ca ta pi ta ca p pi ta ca p sp C tis sp C tis bo t ry tis bo t ry tis bo t ry tis bo t ry bo t ry a al a br al bo gl a al a ep h ac ep h ac ac ep h al a 0.00 c) Brassica napus DFFS set 1 25.00 20.00 15.00 10.00 5.00 A ge n DH om e Sw ed sp r rin a p e g e fo k ra ale g Sp e ra rin pe g Sp O rin SR g Sp OS rin R g Sp O rin SR g Sp OS rin R g Sp OS rin R g Sp O rin SR g O SR sw ed e sw ed e sw ed sw e s e wi de wed nt l er an e fo dra dd ce e W r ra in pe te rO W S in te R W rO S in te R rO W S in te R W rO S in te R rO W S in te R W rO S in te R rO W S in te R rO W S in te R W rO S in te R rO W S in te R W rO S in te R rO SR 0.00 This fatty acid was chosen because selection was not expected to have had much influence on this fatty acid, however, evidence for a significant effect of selection was found. The greatest observed variation was between the accessions in the wild C genome DFFS set with the least variation being found in the B.napus DFFS set. This can be explained due to the effect of strong selection pressure which has been exerted by breeders on the modern B. napus cultivars for high Oleic acid (18:1n9) and SID 5 (Rev. 3/06) Page 6 of 11 low Erucic acid (22:1n9) to meet market needs which presumably has had an indirect effect on the content of 18:3n3; whereas the wild C genome species will not have been subjected to such strong unidirectional selection. It is interesting to note that the domesticated section of the C genome (B.oleracea) displays less variation than the wild species suggesting that some variation may have been lost during domestication. It is clear from the data that we have generated in this preliminary study that there is significant natural variation for both fatty acid profile and amount of individual fatty acids within the seed oil of B. oleracea and its close wild relatives. Objective 2: To identify allelic variation for genes associated with fatty acid biosynthesis A number of genes involved in the biosynthesis pathway of fatty acids have been identified in the model dicotyledon plant and member of the Brassicaceae, Arabidopsis thaliana. These include the fatty acid desaturase genes FAD3 (AT2g29980) and FAD2 (AT3g12120) (Okuley et al., 1994), fatty acid elongases FAE1 (At4g34520) and FAE homologue (At3g52160), palmitoyl-ACP thioesterase FatB (At1g08510) and the acyl carrier protein ACP1 (At3g11430) (Bonaventure and Ohlrogge, 2002). Closely related orthologues of these genes have been isolated from Brassica species. The B. napus genes FAE1.1 and FAE1.2 have been shown to co-segregate with two loci which govern erucic acid production (Barret et al., 1998; Fourmann et al., 1998), with FAE1 genes also being isolated from B. oleracea and B. rapa (Das et al., 2002) as well as B. juncea (Gupta et al., 2004). Between four and six copies of FAD2 have been reported for B. napus (Scheffler et al., 1997) and representative orthologous genes encoding for both FAD2 and FAD3 have been cloned (Arondel et al., 1992; Okuley et al., 1994) In order to amplify locus specific regions of the paralogues of three significant genes involved in fatty acid synthesis (FAE, FAD2 and FAD3) locus-specific primers were designed to an approximately 1000 bp segment of the coding region sequence. These were amplified with a proof-reading PCR polymerase to produce regions of the FAE and FAD genes from a structured set of 188 accessions of the B. oleracea DFFS, and 96 accesions from the C genome DFFS and the B.napus DFFS. The amplification products were sequenced from both strands and the sequences analysed for single nucleotide polymorphisms (SNP’s), synonymous and non-synonymous changes and for the presence of indels which may affect protein function. A significant number of different alleles were identified for each gene. The wild C genome spp DFFS and B oleracea DFFS sets showed a similar range of allelic variants with the B.napus DFFS set exhibiting the least diversity. a) Screening for allelic variants of FAE1-2 A significant number of allelic variants were detected in a Fae_1-2 C genome locus Shown here is variation in sequence found between AA132-148 TTCGGGTCTAGGCGATGAAACCCACGGGCCCGAGGGGCTGCTTCATGTCC ____A_____________________T______________________________.G____ This region showed two synonymous and one non synonymous allelic differences The following illustrates the effect of the above allelic substitutions in the FAE 1-2 gene on the amino composition of part of the protein 134 GLGDETHGPEGLLQVPPRKTFAAAREETE 162 134 GLGDETHGPEGLLHVPPRKTFAGAREETE 162 Non synonymous changes were found at amino acid positions 147, 156,181, 214, 234 in many accessions. Synonymous changes were also often shared between multiple accessions. Additional allelic variants, both synonymous and non-synonymous, were also found which were unique to individual lines. In contrast when the same primers are used to amplify this region of FAE1-2 from the B.napus DFFS only one accession was found to contain an allelic variant. This illustrates the effectiveness of the Brassica oleracea DFFS and the wild C genome DFFS as sources of potentially useful allelic variation which could be introgressed into B.napus. SID 5 (Rev. 3/06) Page 7 of 11 b) Screening for allelic variants of FAD-2 A similar degree of allelic variation was detected within a FAD-2 locus CTCTTCCGTTACGCCGCCGCGCAGGGAGTGGCCTCGATGGTCTGCTTTTACGGAGTC ____A________________________A_____________________________C________ Within the same region 28 accesions had alternative base pair substitutions at AA 247. Substitution sat positions 255 and 261 appeared correlated but had no effect on the AA. 32 out of 96 lines showed nonsynonymous change at AA241. FAD2 AA239-261 GLFRYAAAQGVASMVCFYGVPLLIVN GLYRYAAAQGVASMVCFYGVPLLIVN Outside this region of this FAD2 locus a further 5 SNPs were detected which appeared to be non synonymous. c) Screening for allelic variants of FAD-3 In contrast to FAE1-2 and FAD2 the PCR results with a FAD3 locus suggested that there was no nonsynonymous changes detected. Further work will be required to verify this result. Allelic variants identified by sequence analysis were matched against fatty acid profiles to determine if there were any correlations between sequence variation and long chain fatty acid synthesis. It was difficult to detect any direct relationships due to the number of allelic variants found with no obvious link with the non–synonomous changes in the FAE1-2. However, the results of the preliminary allelic survey for three loci known to affect fatty acid biosynthesis clearly demonstrated allelic variation for genes associated with fatty acid biosynthesis. Objective 3: To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying the gaps in knowledge required to produce that ideotype. There is a large diversity of crop morphology within B. oleracea; there is therefore potential to manipulate crop architecture in order to optimise the crop. In addition there are the tools and resources available in B. oleracea to exploit genetic variation for important input traits such as nitrogen, phosphorous and water use efficiency as well as pest and disease resistance. It is therefore possible to ‘design’ and breed to an ideotype to maximise the sustainability of the crop as a renewable feedstock. The current information relating to vegetable forms of B. oleracea was reviewed this together with information from OSR studies to identify where it is applicable to the development of an oil form of the crop. We also identified where additional information is required. Aspects of the crop which will need to be examined include: Spring sown v autumn sown crops (vernalisation requirement) Canopy structure/branching Flowering time Harvest index Height Root architecture Energy balance/efficiency (input v output) Yield Pod shatter Pest and disease resistance Resource use efficiency Breeding system (hybrid v inbred line) SID 5 (Rev. 3/06) Page 8 of 11 These were considered from a viewpoint of adaptation to climate change, carbon balance, economics, land use, environmental footprint and public perception. An initial brain storming session on the ‘ideal plant’ was carried out; the results of this are given in the appendix. This was the only activity aimed at this objective within the resources of the project. It will need to be taken further and to be informed by results from other projects currently being carried out on OSR and B. oleracea – e.g nitrogen use efficiency LINK project, Phosphorous use efficiency project in OSR and B. oleracea; Novorb LINK project etc. However, it clearly demonstrates the potential of utilising the greater ‘plasticity’ of the C genome (as opposed to the napus genome) to breed a crop with many desirable traits suited to a more sustainable low input production system. Although the C genome contains many gene duplications and triplications the breeding effort required to manipulate plant morphology will be easier in a ‘diploid’ species than in an allotetraploid such as B napus where the degree of gene duplication is doubled. Conclusion: This three month preliminary project had the overall aim to demonstrate the feasibility of developing Brassica alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for the UK. The results obtained clearly indicate that it would be feasible to develop a Brassica oleracea oil crop for the UK. We have demonstrated the existence within the C genome genepool (B. oleracea and related wild species) greater natural variation for seed oil fatty acid profile and seed oil content than was found in the Ac (B. napus) genepool. We have also demonstrated allelic variation for genes known to be associated with fatty acid biosynthesis in the C genome genepool. This provides the underpinning basis for developing an oil crop in which cultivars are bred to produce oils tailored for specific industrial end uses. The ‘plasticity’ of the C genome as demonstrated by the many and varied crop morphologies within the species and also the variation in morphology between the wild relatives provides the genetic basis for redesigning a crop ideotype to provide a new oil crop which meets stakeholder needs in terms of agronomy, yield and reduced (potentially beneficial) environmental impact. The project has therefore clearly demonstrated the feasibility and potential of developing a form of Brassica oleracea as a new industrial oil crop for the UK. It has also clearly demonstrated that the C genome genepool is a source of potentially useful genetic variation for improving the UK OSR crop. However, there is a need for a more extensive future project to provide the underpinning knowledge to undertake either of these routes. Future work: A future project would exploit the range of experimental genetic and genomic resources in C genome brassica available at Warwick HRI. We are currently developing the Diversity Sets (DS) for B. oleracea and wild C genome Brassica spp related to B. oleracea. These unique structured samples of the genetic resource collections would be used to extend the current study of the natural genetic variation within the C genepool at both the phenotypic level and the level of allelic variation for ‘key’ genes in oil biosynthesis. Warwick HRI (W HRI) also maintains a number of defined Brassica mapping populations of double haploid (DH) and substitution lines under both the OREGIN (Defra) and AdVaB (BBSRC Crop Sci) projects. (QTL analysis using two of our B.oleracea mapping populations have already identified 22 QTL associated with metabolic step changes in FA synthesis). A wide selection of brassica genomic libraries (EST, Genomic and BACs) are available at W HRI and considerable effort has been put into adding value to these though physical mapping (BBSRC, IGF Project), hybridisations of macroarrays and BAC end sequencing. W HRI is also directly involved in the multinational Brassica genome sequencing programme (MBGP) funded through BBSRC. We are therefore in a position to move from the identification of QTL to the identification of the genes responsible for the traits of interest. Carrying out these studies in B oleracea avoids the problems associated with the additional level of gene duplication in tetraploid B. napus. We will use this knowledge in conjunction with our SID 5 (Rev. 3/06) Page 9 of 11 knowledge of the pathways involved in FA synthesis and catabolisim, to design a pre-breeding program to combine alleles to provide a FA profiles matching those identified by chemists and materials engineers as optimal for different end uses. In addition we will also exploit our existing knowledge of the genetics of seed size and oil content and other components of yield to maximize the energy embodied in the oil. The C genome is also a strategic source of beneficial alleles for traits with an environmental benefit. We have already demonstrated the value of the B. oleracea diversity set in studies to assess variation at the phenotypic level for traits such as phosphorous use (PUE) and water use efficiency (WUE). We hypothesise that the C genome wild species DS is a potentially rich source of beneficial alleles for ‘traits for sustainable production’ as these species have evolved under natural low input conditions. We will survey for allelic variation at the sequence level in key genes associated with traits for sustainability with the aim of identifying allelic variants associated with phenotypic variation in order to provide beneficial alleles for breeding for increased sustainability of production. This approach will increase the efficiency of identifying potential beneficial alleles since only those accessions possessing allelic variants need to be phenotyped. We have initiated a demonstration of this approach within the OREGIN project with an allelic survey of a subset of the B. napus DS for one of the cytoplasmic isoforms and one of the plastidic isoforms of glutamine synthetase. However, this study has shown the difficulty of attempting to identify allelic variation in an allotetraploid. We will therefore use a strategy of initial gene identification and function studies in the genetically less complex diploid species rather than the allotetraploid. All of the species are compatible with B. oleracea and so novel allelic variants can be introgressed into the domesticated genepool either directly to vegetable brassicas or via inter-specific hybridisation and embryo rescue to OSR. We will deliver the outputs of this ‘pre breeding’ programme to the oil seed breeding community by introgressing them into a common ‘crop portal’. This would be a DH line(s) derived from a well adapted cultivar(s) to be identified by the OSR group of the British Society of Plant Breeders. SID 5 (Rev. 3/06) Page 10 of 11 References to published material 9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project. Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR (1992) Map-Based Cloning of a Gene Controlling Omega-3-Fatty-Acid Desaturation in Arabidopsis. Science 258: 1353-1355 Barret P, Delourme R, Renard M, Domergue F, Lessire R, Delseny M, Roscoe TJ (1998) A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid. Theoretical and Applied Genetics 96: 177-186 Bonaventure G, Ohlrogge JB (2002) Differential regulation of mRNA levels of acyl carrier protein isoforms in Arabidopsis. Plant Physiology 128: 223-235 Das S, Roscoe TJ, Delseny M, Srivastava PS, Lakshmikumaran M (2002) Cloning and molecular characterization of the Fatty Acid Elongase 1 (FAE 1) gene from high and low erucic acid lines of Brassica campestris and Brassica oleracea. Plant Science 162: 245-250 Fourmann M, Barret P, Renard M, Pelletier G, Delourme R, Brunel D (1998) The two genes homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in Brassica napus. Theoretical and Applied Genetics 96: 852-858 Gupta V, Mukhopadhyay A, Arumugam N, Sodhi YS, Pental D, Pradhan AK (2004) Molecular tagging of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide polymorphisms in FAE1 gene. Theor Appl Genet 108: 743-749 Larson TR, Graham IA (2001) Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant J 25: 115-125 Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J (1994) Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6: 147-158 Scheffler JA, Sharpe AG, Schmidt H, Sperling P, Parkin IAP, Lühs W, Lydiate DJ, Heinz E (1997) Desaturase multigene families of <SMALL>Brassica napus</SMALL> arose through genome duplication. TAG Theoretical and Applied Genetics 94: 583 SID 5 (Rev. 3/06) Page 11 of 11