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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 IF0168 Improved resolution of QTL associated with Water and P use efficiency 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 6 £ 49,810 start date ................ 01 October 2008 end date ................. 31 March 2009 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. Water is the most important factor limiting crop production. Crop production in the UK is either rain-fed, as in the case of the majority of arable crops, or relies on supplementary irrigation. The total amount of water abstracted for irrigation of field crops is around 130 million cubic meters (Mm 3), with protected edible crops and ornamentals using approximately another 60 Mm 3 for irrigation. Water availability during the UK growing season is predicted to decline and may make some non-irrigated crop production economically unsustainable. In addition the excessive use of soil water by non-irrigated crops reduces the amount of rain water returned to surface or ground water reserves. Irrigated crops will also require a greater input of supplementary irrigation to maintain production because of both reductions in rain-fall and increases in evapotranspiration as average temperatures increase. Consequently, there is a need to minimise the amount of water required to grow crops, and to this end it will be important to have crop varieties that can use the available water more efficiently. In addition to water, most crops require high inputs of inorganic fertilisers to maintain yield and quality. These fertilisers are energy intensive to produce and can (if not correctly managed) pollute adjacent environments leading to a loss of biodiversity. Capture of nutrients from the soil is a key crop trait that impacts on the quantity of fertilizer that needs to be applied to soil and the entry of nutrients into water courses. Development of crop varieties that can maintain yields with lower fertiliser inputs and that recover more applied nutrients are key to the development of low input agricultural systems. Previous work (HH3501, WQ0119, HH3608TX, LK0979, IF0125, HH3608TX, WU0116), has identified QTL associated with water-use efficiency in the Brassica oleracea genome and validated this in the substitution line AGSL118, which differs from the recurrent parental line (A12DHd), by substitutions of genomic DNA from the other parent (GD33) in linkage group, C1, C6 and C7. Several QTL have also been identified for various measures of phosphorus use efficiency (HH3501) on linkage groups C3 and C7 of B. oleracea in a number of substitution lines. In order to define the position of these QTL more precisely, back-crosses to the A12 parent have been performed and stocks of F1 or F2 seed produced, depending on the lines. For example, previously AGSL118 was crossed to A12, and the resulting line (BC 1F1) was grown and allowed to self-pollinate to give the BC1F2 seed stock. The aim of this project was to identify recombinants in the BC 1F2 population of the cross A12 x AGSL118 using molecular markers. A panel of homozygous recombinant lines would then be assessed for phenotype to establish a single small mapping interval for each QTL. SID 5 (Rev. 3/06) Page 2 of 6 This current project had two objectives to facilitate the continued back-crossing of these lines to fine map QTL associated with water and phosphorus use efficiency. In Objective 1, a total of 421 BC1F2 plants from the introgression line AGSL118 were sown in the glasshouse and sampled to obtain DNA. PCR amplifications were made on these DNA samples using the primer pairs for markers identified as close as possible to the proximal and distal ends of the introgressed regions in line AGSL118. In initial screens, two classes, either single or double introgressions, of plant genotype were selected and retained for self pollination and seed production. Of these approximately 70 of these plants, representing all genotypes, have been grown to flowering and BC1F3 seed will be collected within two months. In Objective 2, a total of 17 potential markers spanning the map interval of the introgressed regions for AGSL118 were screened on GD33 and A12DHd parental and BC1F1 (heterozygous AGSL118) DNA. Three of these markers were associated with linkage group 1, five with linkage group 6 and nine with linkage group 7. Four markers shown to be polymorphic between the two parents and within the introgressed GD33 regions of AGSL118 were selected for screening our BC1F2 plants. 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). Background Water is the most important factor limiting crop production. Crop production in the UK is either rain-fed, as in the case of the majority of arable crops, or relies on supplementary irrigation. The total amount of water abstracted for irrigation of field crops is around 130 million cubic meters (Mm 3), with protected edible crops and ornamentals using approximately another 60 Mm 3 for irrigation. Water availability during the UK growing season is predicted to decline and may make some non-irrigated crop production economically unsustainable. In addition the excessive use of soil water by non-irrigated crops reduces the amount of rain water returned to surface or ground water reserves. Irrigated crops will also require a greater input of supplementary irrigation to maintain production because of both reductions in rain-fall and increases in evapotranspiration as average temperatures increase. Consequently, there is a need to minimise the amount of water required to grow crops, and to this end it will be important to have crop varieties that can use the available water more efficiently. In addition to water, most crops require high inputs of inorganic fertilisers to maintain yield and quality. These fertilisers are energy intensive to produce and can (if not correctly managed) pollute adjacent environments leading to a loss of biodiversity. Capture of nutrients from the soil is a key crop trait that impacts on the quantity of fertilizer that needs to be applied to soil and the entry of nutrients into water courses. Development of crop varieties that can maintain yields with lower fertiliser inputs and that recover more applied nutrients are key to the development of low input agricultural systems. Previous work (HH3501, WQ0119, HH3608TX, LK0979, IF0125, HH3608TX, WU0116), has identified QTL associated with water-use efficiency in the Brassica oleracea genome and validated this in the substitution line AGSL118, which differs from the recurrent parental line (A12DHd), by substitutions of genomic DNA from the other parent (GD33) in linkage group, C1, C6 and C7. Several QTL have also been identified for various measures of phosphorus use efficiency (HH3501) on linkage groups C3 and C7 of B. oleracea in a number of substitution lines. SID 5 (Rev. 3/06) Page 3 of 6 In order to define the position of these QTL more precisely, back-crosses to the A12 parent have been performed and stocks of F1 or F2 seed produced, depending on the lines. For example, previously AGSL118 was crossed to A12, and the resulting line (BC1F1) was grown and allowed to self-pollinate to give the BC1F2 seed stock. The aim of this project was to identify recombinants in the BC1F2 population of the cross A12 x AGSL118 using molecular markers. A panel of homozygous recombinant lines would then be assessed for phenotype to establish a single small mapping interval for each QTL. Results and Discussion Objective 1. Continue back-crossing Brassica oleracea lines to further reduce the size of substitutions and improve the resolution of recently discovered QTL for water and phosphorus-use efficiency. The line AGSL118 has lower WUE and PUE than A12, and thus the three introgressions (on C1, C6 and C7) in this line must contain a locus or loci that control these traits. We aimed to separate the three introgressions and screen for recombination within the introgression in linkage group 7. A total of 421 BC 1F2 plants from the introgression line AGSL118 were sown in modules in the glasshouse. Ten plants of the parental line A12DHd were also sown with each batch. Plants were sampled in batches of 96 for DNA extraction from the 1 st true leaf at the 2nd true leaf stage (approx 4 weeks after sowing). DNA was extracted in 96 well format by homogenization of leaf material in a tissue mill, followed by extraction using a commercially available kit (Qiagen DNeasy 96). Extracted DNA was stored at -20oC. PCR amplifications were made in 96 well format using the primer pairs for markers identified as close as possible to the proximal and distal ends of the introgressed regions listed under Objective 2. Fluorescently labelled reverse orientation primers were used to facilitate capillary electrophoresis genotyping (Fig 1). PCR amplifications for each primer pair were performed separately and aliquots of the amplifications then pooled for each sample to allow for screening of multiple markers from a single plate. In initial screens, two classes of plant genotype were selected and retained for self pollination and seed production (Table 1). 1) Single introgressions, where the 3 introgressions present in AGSL118 had been reduced down to just one, either in the homozygous or heterozygous form. Four recombinants in the linkage group 7 introgression have also been detected, in a background lacking the linkage group 1 and 6 introgressions, offering an opportunity to begin resolving the region of interest. 2) Double introgressions, where combinations of 2 of the 3 AGSL118 introgressions have been inherited. These lines will enable us to test for allele combination effects should the phenotype not be retained by any of the three single introgression lineages. Figure 1. Automated genotyping traces corresponding to heterozygote signals for two of the markers screened; Ni4B10 (175bp and 192bp) and sora93 (445bp and 479bp). The A or G suffix indicates the trace associated with either the A12DHd allele (A) or the GD33 allele (G). SID 5 (Rev. 3/06) Page 4 of 6 Table 1. Numbers of AGSL118 plants detected (from 421 individuals) in each of the genotypic categories identified. G and A indicate homozygous GD33 and A12DHd genotypes respectively. H indicates a heterozygous genotype. Genotypes Linkage Linkage Linkage Number of Plants Group C1 Group C6 Group C7 Single G A A 3 Introgression H A A 3 G A A 1 H A A 11 G A A 10 H A A 9 Double G G A 1 Introgression H G A 7 G H A 8 G G A 4 H G A 8 G H A 6 G G A 3 G H A 17 H G A 8 Total number of plants 99 Approximately 70 of the above plants, representing all genotypes, have been grown to flowering and BC 1F3 seed will be collected within two months. Objective 2. Develop new markers for improving the resolution of QTL associated with water and phosphorus use efficiency Based on the existing B. oleracea genetic map, a total of 17 potential markers spanning the map interval of the introgressed regions for AGSL118 were screened on GD33 and A12DHd parental and BC 1F1 (heterozygous AGSL118) DNA. Three of these markers were associated with linkage group 1, five with linkage group 6 and nine with linkage group 7. Four markers shown to be polymorphic between the two parents and within the introgressed GD33 regions of AGSL118 were selected for screening our BC 1F2 plants. (Table 2). Table 2. Results of marker screening on GD33DHd, A12DHd and AGSL118 DNA. ? indicates that further optimisation of the PCR assay is required in order to obtain conclusive results. Markers selected for screening are highlighted in italics. Size (bp) Linkage Map position Within introgressed Group Marker (cM) region? GD33 A12 1 mSN3734a 65 No 288 1 Ol10F11 66 No 148 1 Ni4B10 101 Yes 192 175 6 6 6 6 6 mCRABSCLAWa 028L01AT3a BoAP1-c mBRMS298a Ca72 38 39 40 41 82 No ? ? No Yes ? ? 232 234 273 ? ? 219 253 7 7 7 7 7 7 7 7 7 sore90a_125 mBRAS023a_11 mBRAS019a_11 mNA12F03a mCB10299a mBRMS296a mFITO222c mA48350b sora93 ? 3 4 8 12 12 14 14 15 No Yes Yes Yes ? ? Yes Yes Yes 206 151 312 139 ? 409 263 479 128 197 114 297 139 ? 385 259 445 SID 5 (Rev. 3/06) Page 5 of 6 For linkage group C7 we were able to select two informative markers, one at either end of the introgressed region, allowing us to detect recombinants within the interval. There are four more markers immediately available to further characterise the selected plants for this C7 region. Only one of the 5 selected C6 markers was found to lie within the introgressed GD33 region of AGSL118 when genetically tested. This one marker, Ca72, has allowed us to make informed selections at his stage; however, to allow further characterisation of this region the development of more suitable markers is required. Only one of the 3 selected C1 markers was found to lie within the introgressed GD33 region of AGSL118 when genetically tested. This one marker, Ni4B10, has allowed us to make informed selections at his stage. However, it is noteworthy that the C1 introgressed region of AGSL118 is largely in common with the introgressed region of AGSL101, currently being characterised by Prof. Bill Finch-Savage as part of a BBSRC funded project (BB/E006418/1). The polymorphic markers and BAC sequence data developed in this BBSRC project could be directly applicable, should phenotypic characterisation of lines with single introgressions point towards C1 as containing our gene(s) of interest. Future work For AGSL118, lines containing single and double introgressions for the linkage groups 1, 6 and 7 now need to be assessed for WUE and PUE. This will determine the introgression, or combination or introgressions, responsible for the WUE and PUE effects. In order to fine-map the QTL, recombinations within that region need to be selected, and phenotyped. Very closely linked markers would become tools suitable for brassica breeding programs. Such markers could also then be placed on the physical map, and finally the causative gene(s) identified by further marker discovery and fine mapping. Once a causative gene is known, it would become a breeding target in non-brassica crops. Similar work is required for other substitution lines, where the work is less advanced, e.g. where we now have generated BC1F2 seed suitable for mapping of PUE effects within specific introgressions. Again, this will lead to genetic markers that could be used to select for increased PUE in brassica species. 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. N/A SID 5 (Rev. 3/06) Page 6 of 6