General enquiries on this form should be made to: Defra, Procurements and Contracts Division (Science R&D Team) Telephone No. 0207 238 5734 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 Project identification of its completed research projects in the public domain wherever possible. The GC0140 SID 5 (Research Project Final Report) is 1. Defra Project code designed to capture the information on the results and outputs of Defra-funded 2. Project title research in a format that is easily Fingerprinting the National Apple & Pear Collections publishable through the Defra website. A SID 5 must be completed for all projects. This form is in Word format and the boxes may be expanded or reduced, as 3. Contractor East Malling Research appropriate. organisation(s) New Road East Malling ACCESS TO INFORMATION Kent The information collected on this form will ME19 6BJ 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 54. Total Defra project costs £ 319,372.00 project. Defra may also disclose the (agreed fixed price) information to any outside organisation acting as an agent authorised by Defra to 01 April 2007 process final research reports on its 5. Project: start date ................ behalf. Defra intends to publish this form on its website, unless there are strong end date ................. 31 March 2010 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. 05/09) Page 1 of 18 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. Aims and Objectives Defra holds the National Fruit Collections (NFC) sited in Brogdale (Kent) and curated scientifically by the University of Reading. These collections constitute valuable genetic resources for the genetic improvement of the UK's principal fruit crops They contain approximately 2200 apple and 560 pear accessions, including dessert, culinary, ornamental and cider/perry types, as well as collections of other fruit crops. In recent years, the use of DNA markers for characterisation of germplasm collections has become increasing common. The markers most commonly used, known as Simple Sequence Repeats (SSRs), are areas of the genome of repetitive sequence (e.g. AT AT AT AT AT AT) and variable length (eg. „AT AT AT‟ vs „AT AT AT AT‟). These variations in length can be detected in the laboratory and compiled to create a genetic „fingerprint‟ of an individual tree. Such fingerprints are invaluable aids to the management of collections, eg when checking for trueness to type after propagation or for detecting likely duplicates. In addition, the determination of incompatibility (S) genotype by molecular methods is proving useful for fingerprinting Rosaceous tree fruits as well as providing agronomically useful data. Continuing the work started in Defra funded project GC0139, this proposal aim to complete the characterisation of the pear collection and similarly characterise the apple accessions. The main objectives were to: . Use microsatellites to fingerprint the apple accessions in the National Fruit Collection 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. Complete the data set of microsatellite fingerprints of the pear accessions in the National Fruit Collection 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. The work was broken down into the following technical aims: 1. to extract DNA samples from the accessions of the apple collection and the remaining half (275) of the pear collection 2. to optimise PCR conditions for ~12 informative microsatellite primers in apple, developing multiplexes if appropriate 3. to determine the microsatellite fingerprints of the accessions of the apple collection and the remaining half of the pear collection 4. to verify ploidy levels of all accessions which appear from microsatellite analysis not to be SID 5 (Rev. 05/09) Page 2 of 18 diploid 5. to collate the data into Excel spreadsheets and provide to the scientific curator, e.g. to allow the search for duplicates, to submit the data to the freely-accessible ECPGR Malus and Pyrus databases and to produce papers on ploidy and fingerprinting. Main findings A total of 559 pear and 2,162 apple accessions were analysed with twelve SSRs chosen from the marker sets recommended by the ECPGR for each genus. A set of eight control genotypes for each crop were also included in the analysis to allow for the internal harmonization of data and to aid comparison of results with other studies. Analysis of the SSR data identified a total of 443 and 1,613 unique accessions of pear and apple respectively with the remaining individuals having at least one other accession with identical SSR profile. A total of 43 and 193 groups of suspected duplicates were identified in pear and apple respectively and they are presented in Tables 2 & 3. Some of these groups are made out of the known clones of popular cultivars - e.g. 15 „Williams‟, 7 „Conference‟ and 5 „Comice‟ clones in the case of pear and 20 „Jonagold‟, 21 „Golden Delicious‟ and 20 „Cox‟s Orange Pippin‟ clones in the case of apple were identified - whilst other groups could indicate previously unknown and/or unwanted replication, mislabelling etc. All data generated from this project has been tabulated and sent to Curator of the National Fruit Collection where morphological data will be used to determine if the accessions within the groups are in fact identical. These fingerprints will prove to be an extremely valuable reference set for testing the trueness-to-type of the recently re-propagated pear collection and the soon to be re-propagated apple collection. The re-propagated accessions fingerprinted using the same methodology used in this study and the two data sets compared – thus avoiding in most cases the need for laborious and time-consuming morphological comparison. Additionally, 2,095 accessions were analysed using the consensus primers to amplify alleles of the S-locus and twenty-two putative new alleles were identified. At least one allele was amplified for each accession and 1,696 genotypes were fully characterised. Unfortunately, 399 accessions remained not fully resolved with one or more of their incompatibility alleles still undetected. Further research will be needed to confirm the new alleles identified and to improve the methodology in order to fully characterise the S genotype of all accessions. Apples and pears are generally diploid (their cells contain two copies of each chromosome) however certain cultivars are polyploid i.e. they present three or four copies of each chromosome. Cytometric analysis was undertaken to confirm ploidy levels where SSR analysis indicated that an accession could be polyploid. A total of 48 pear and 304 apple accession have been confirmed as polyploids (Tables 6 & 7). Future work and prospects EMR has been recently commissioned to fingerprint two other UK fruit collections and result from these analyses will be compared to the data for the NFC. This would allow rationalising the germplasm kept in these other collections and present new candidates for accession into the NFC. As other international groups adopt the harmonised fingerprinting protocols for apple and pear it will be possible to compare data between collections which could lead to the identification of accession errors and even to the rationalisation of germplasm collections across Europe. Furthermore, different species of Pyrus and Malus evolved in a wide range of environmental conditions are known to have contributed to the current range of cultivated pears and apples respectively.