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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.
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.
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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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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
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.
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). Scientific objectives of the contract To use microsatellites to fingerprint 275 pear accessions in the National Fruit Collections and provide curators with a valuable data set which distinguishes clearly all or most of the varieties tested thus enabling checking of identities and detection of synonyms Extent to which the objective set out in the contract was met The objective was met in full and, in fact, exceeded. In all, 276 accessions were fingerprinted not 275. DNA was extracted from the 276 accessions; the extracts were amplified with a set of thirteen microsatellite primers that were agreed at the ECPGR workshop attended by international experts; the amplification products were separated on an automated sequencer; and the genotypes recorded into an Excel spreadsheet that was provided to the scientific curator at Imperial College. Materials and methods Technical aim 1- DNA extraction Leafy shoots were collected from one of each pair of accessions of the pear collection at Brogdale by the Sub-contractor and were labelled. Leaves were removed, frozen in liquid nitrogen and stored in a -80oC freezer at EMR. DNA was extracted from half the samples (276 accessions) using 0.2g tissue following a modified CTAB protocol (James et al. 2003). Leaves were also collected and DNA extracted from the eight Brogdale accessions selected as controls at the ECPGR workshop organised by EMR. The extracts were quantified and partially qualified by electrophoresis through agarose. The quality of the DNA was further assessed by checking the amplification the samples with a fully optimised PCR. A full list of the accessions characterised can be found in Appendix 1. Technical aim 2 - Choice of primers and optimisation of PCR conditions The choice of a set of microsatellites for fingerprinting pears was discussed at the recent ECPGR workshop attended by international experts. Markers have been chosen from each linkage group (where possible) that are robust, preferably single locus and that have been proved to be polymorphic in previous studies. From those recommended, we chose 13 that, fluorescently labelled, were combined into four ‘multiplex’ combinations (see Appendix 2). The most informative 12 primers that were chosen as the ECPGR core set (priority groups 1, 2 and 3 in the table) were optimised into three multiplexes for future use. PCR conditions were optimised to obtain robust and reliable amplification. Primer 13 (CH_Vf1) consistently produced a ‘third’ peak which could not be ignored even though we are confident from the rest of the results that the accessions involved are not triploids. Technical aim 3 - Fingerprinting the pear collection The DNA samples were amplified with the chosen primers, in multiplex reactions, using a thermocycler machine and ensuring the presence of the selected control samples within each plate. The amplification products were loaded on to an ABI automated sequencer so that the alleles could be sized. Then the data were compiled using GENESCAN and GENOTYPER software. The quality and reliability of the fingerprints was assured by using standardised methodologies with optimised primers and regular control samples. Hard copies of all traces from the software were printed allowing clear comparisons between peak intensity and fragment size of the amplified products. Any variation in peak size between plates was normalised by comparing the control samples. All data were checked twice by two individual staff members and repeats were performed on any sample where the traces were unclear. Data were stored within EMR’s EMQA system. Technical aim 4 - Collating genotypes An Excel spreadsheet was prepared giving the genotypes at each microsatellite locus of the pear accessions. This has been provided to the scientific curator at Imperial College. Data have been analysed using GenAlex and PAUP* software to generate dendrograms to help identify duplicates in collaboration with Sub-contractor. The Pyrus database manager has been notified that the data set is now available for inclusion. A draft paper has been prepared for the Eucarpia Fruit Breeding Symposium to be held in September 2007. Results Comparisons between the traces have revealed several groups that are indistinguishable by the SSRs used. These are listed in Appendix 3. Some of the names reveal that these are in fact clonal cultivars which we would expect to appear identical using this method. Discussion of the results and their reliability Discussion: Information regarding the optimised multiplexes has been distributed to other European groups who have already adopted their use. This should allow comparison of data sets between collections e.g. to confirm identities and, perhaps, reduce duplication. Although the data from primer 13 (CH_Vf1) is included in this report, they have been dropped from the ECPGR priority core set. Our data set should assist the curator of the pear collection to confirm some duplicated material. Identification of pairs/groups with indistinguishable SSR fingerprints, combined with morphological observations should enable decisions to be made about possible future de-accessions from the collection. Reliability: We are confident about the reliability of these results. The initial leafy samples were collected by Dr Alison Lean who has worked with the National Fruit Collections for over 10 years. All samples were labelled clearly with a unique identifier code which relates to the physical position of each source tree within the Collection. The quality and reliability of the fingerprints was assured by using standardised methodologies with optimised primers and regular control samples. Hard copies of all traces from the software were printed allowing clear comparisons between peak intensity and fragment size of the amplified products. All data were checked twice by two individual staff members and repeats were performed on any sample where the traces were unclear. Main implications of the findings The fingerprinting will be a significant aid to the efficient management of the collections, as has been demonstrated by the cherry pilot project, allowing some duplicates to be detected. The data will be a useful reference should the recently re-propagated pear collection be fingerprinted to compare against the original collection. The traditional method of doing this, using morphological comparison, is time consuming and takes several years. The agreement at European level on standard microsatellites and reference control cultivars is a major advance and makes this work of international, as well as national, relevance. Possible future work The first priority for future work is to complete this fingerprinting exercise with the rest of the pear collection. This data set would identify further duplicates and would complete the reference data set. Fingerprinting the recently re-propagated pear collection would allow the newly propagated trees to be verified against the established collection. There is scope for characterising the pear collection for features relevant to climate change e.g. flowering time or winter chill, by molecular and phenotypic approaches, and, indeed, using the available genetic resources to investigate the genetic control of such important traits. Action resulting from the research The primary action from this research is to enable the curator of the National Fruit Collections to identify and remove duplicates. As the ECPGR initiative to suggest a core set of microsatellites for fingerprinting fruit collections coincided with this project, the reference data set that we have presented to the ECPGR Pyrus database is the first data set of this size. Initiation of this work has helped to promote this co-ordinated movement internationally, with interest from both New Zealand and the U.S.A. Presentations and industry interaction This project was briefly described during discussions at the ECPGR microsatellite workshop at East Malling Research in December 2006 and at the National Fruit Collections Advisory Committee meeting in January 2007. Some of the data will be presented at the Eucarpia Fruit Breeding Symposium in September 2007 and at the planned EMRA ‘DNA day’ at EMR in November 2007. Appendix 1: List of the accessions characterised
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