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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 HH3717STF Extending the season of stone fruit by breeding lateripening cherries and medium-early and medium-late plums Contractor organisation(s) East Malling Research New Road East Malling, Kent ME19 6BJ 54. Total Defra project costs (agreed fixed price) 5. Project: Page 1 of 18 £ 490,000 start date ................ 01 April 2003 end date ................. 31 March 2008 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 UK market for stone-fruit is buoyant, but there are unfilled gaps in supply regarding early plums and late cherries. East Malling Research (EMR) has a wealth of breeding lines, intraspecific and interspecific, and expertise in genetics and markers that was used for the genetic improvement of these two crops. The objectives of the project were: 1. To breed and select large-fruited, late-ripening cherry selections for grower-funded trial 2. To introgress pest and disease resistance from interspecific breeding lines into sweet cherry 3. To raise interspecific progenies of sweet cherry at tetraploid level 4. To identify and apply molecular markers to benefit of cherry improvement programme 5. To breed and identify large-fruited red plum selections ripening between ‘Opal’ and ‘Victoria’ or between ‘Victoria’ and ‘Marjorie’s Seedling’ for grower-funded trial 6. To transfer technology to the industry 7. To review progress of the project Objective 1 About 500 intraspecific cherry seedlings per annum in the fruiting assessment plots were netted and assessed for crop,season and fruit quality as well as tree habit and field resistance to canker, Promising individuals, 37 in all – ideally with spreading tree habit and canker resistance and having large, firm, well-flavoured, late black fruit resistant to cracking – were selected for trial and propagated on to Gisela rootstock to raise trees for industry-funded trial. Fourteen selections were chosen in 2003 (and subsequently planted in the HDC-funded variety trial MP183 in March 2005) – C216-3 (C3-5 × ‘Lapins’), C247-1 (‘Colney’ × ‘Sunburst’), C250-1 and SID 5 (Rev. 3/06) Page 2 of 18 2 (parentage uncertain), C261-52 (‘Merton Late’ × ‘Colney’), and C273-1 and -11 (‘Colney’ × ‘Sweet September’), C304-35 (‘Stark Hardy Giant’ × ‘Colney’), C308-4, -20, -64, -68 and OA (‘Colney’ × ‘Gaucher’) and C318-30 (‘Colney’ × ‘Stark Hardy Giant’). Seven selections were chosen in 2004 (and subsequently planted in HDC-funded variety trial MP187 in March 2006) – C328-1 and -22 (‘Summit’ × ‘Strawberry Heart’), C338-3 and -14 (‘Stella’ × ‘Van’) and C358-1, -2 and -4 (‘Ferbolus’ × ‘Penny’). Nine selections were chosen in 2005 and 2006 (and are available for planting in March 2008) – C336-10 (‘Lapins’ × ‘Van’), C365-1 (‘Stark Hardy Giant’ × ‘Colney’), C367-9 (‘Ferbolus’ × ‘Colney’), C369-5 (‘Summit’ × ‘Colney’), C371-4 (‘Colney’ × ‘Sunburst’), C374-5 (‘Colney’ × ‘Bing’), C417-30 and -92 (‘Colney’ × C210-7) and C421-03 (‘Vic’ × ‘Colney’). Of these, C367-9 was particularly interesting – remarkably resistant to rain-induced cracking in 2005 and fruit weighing up to 16 g. And seven selections were chosen in 2007 (and will be available for planting in March 2009) – C445-17 (‘Sylvia’ × ‘Noir de Meched’), C454-2 (‘Sunburst’ × ‘Noir de Meched’), C458-1 (‘Penny’ × A53), C478-3 (‘Sasha’ × ‘Lapins’), C499-3 (‘Sweetheart’ × C273-18), C512-6 (C18-19 × ‘Colney’) and C417-317 (‘Colney’ × C210-7). Among progenies of genetic interest were those of ‘Donnissens Gelbe’ × ‘Alman Gulrod’ and ‘Donnissens Gelbe’ × ‘Victor’ which both segregated ~2:1 for green versus yellow-green young leaves and the backcross ‘Decumana’ × C249-8 which segregated ~1:1 for normal versus tobacco-like leaf. To raise additional seedlings for evaluation, various crosses were made under glass, using as parents eg C210-7, C210-18, ‘Canada Giant’, ‘Noir de Meched’, ‘Octavia’, ‘Penny’, ‘Regina’, ‘Sasha’, ‘Summersun’, ‘Summit’ and ‘Sunburst’. Objective 2 About 200 interspecific hybrids in the field deriving from crosses of sweet cherry with P. incisa and P. nipponica, which are resistant to blackfly and/or leafspot, were netted and assessed for fertility and fruit characters. Many were infertile but some cropped well and 20 were propagated on rootstocks. These were tested for resistance to blackfly by controlled inoculation in the glasshouse. Selections C372-1 (‘Colney’ × C57-38) was the most resistant. Seedlings from the cross of ‘Napoleon’ × P. nipponica were tested for resistance to leafspot by a collaborator in Germany. Further crosses were made under glass between, e.g., C57-46 and good black-fruited sweet cherries and the resulting seeds sown. Objective 3 Tetraploid versions of the sweet cherries ‘Stella’ and ‘Summit’ that had been created in a previous project were worked on to rootstocks to raise trees for use in crossing. In the final year, some flowers were produced but the quantity of pollen was slight and no fruit resulted on that occasion from crosses onto naturally tetraploid sour cherry. Objective 4 To develop microsatellite markers of cherry origin for use in fingerprinting and mapping, clones from a genomic library of ‘Napoleon’ were screened for simple sequence repeats and primers designed to flank the repeat sequences; 21 primer pairs were thus developed. The microsatellite map based on a progeny from the cross of ‘Napoleon’ × Prunus nipponica that had been initiated before the project commenced was completed for publication. The seedlings were analysed for some 160 microsatellites and from the cosegregation data a linkage map was constructed with JOINMAP software. The map spanned 680 cM over eight linkage groups with an average spacing of 3.9 cM/marker. A set of 20 microsatellite markers from the ‘Napoleon’ × P. nipponica map that were well distributed across the Prunus genome was analysed in a small progeny of seedlings from the cross ‘Bradbourne Black’ (black) × ‘Merton Late’ (white) that segregated for flesh colour and in this way the Bf gene for black flesh was located on linkage group 3. Further analysis, in a progeny from the cross ‘Napoleon’ (white) × ‘Merton Heart’ (black) located Bf very close to microsatellite AMPA119. In addition, the locus for yellow skin colour, ys, a recessive character segregating approximately 1:1 in the progeny of ‘Donnissens Gelbe’ (yellow skin) × ‘Alman Gulrod’ (blush), was mapped to linkage group 3. Work to understand the nature of the self-compatible alleles S3’ and S4’, which are of considerable agronomic importance as self-compatible cultivars can be grown without pollinators, was completed. Sequencing of the SFB genes of these pollen-part mutants showed that the first is due to a deletion of the SFB gene and the second to a premature stop codon causing the SFB protein to be truncated. And a novel form of self-compatibility in the Sicilian sweet cherry SID 5 (Rev. 3/06) Page 3 of 18 cultivar ‘Kronio’ was characterised and attributed by crossing tests and sequencing to a natural pollen-part mutant of allele S5, denoted S5'. Objective 5 Some 300 plum seedlings, mostly from crosses made in 1997 and 1998, growing in the seedling selection plots were assessed for season, yield, fruit colour, size and eating quality. In March 2007 two early selections, P6-19 (‘Opal’ × ‘Herman’) and P7-38 (‘Opal’ × ‘Avalon’), were benchgrafted on St Julien A to raise trees for trial. Various crosses were made under glass, using ‘Avalon’ and ‘Excalibur’ (excellent quality, red colour), ‘Jojo’ (resistant to plum pox, self-fertile), ‘Jubileum’ (good quality, self-fertile), ‘Opal’ (good quality, early), ‘Reine Claude de Bavay’ (excellent quality, self-fertile), ‘Victoria’ (self fertile, good quality, red colour), ‘Voyager’ (good quality, self-fertile) and ‘Anita’ (exceptionally large fruit). The resulting seedlings were potted up. Objective 6 Thirty-seven cherry selections and two plum selections were propagated for, or planted in, industry-funded trial. Accounts of the programme were given to growers and advisors at Members Days and Fruit Walks. The incompatibility genotypes of 15 advanced selections in trial were determined using primers developed during the project so that appropriate pollinators could be recommended. Likewise, the incompatibility genotypes of the 200 sweet cherries in the Brogdale collection were determined. And, in addition, these cultivars were fingerprinted with 13 microsatellite primers, detecting duplicates and providing valuable data for checking trueness-to-type. Ten non-refereed articles and 21 refereed papers were published or submitted during the course of the project and 31 presentations made to growers, amateurs or scientists. 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). Introduction and objectives The UK acreage of cherry (Prunus avium) and plum (Prunus domestica) is approximately 1700 ha. The wholesale value of the crop fluctuates from about £8M to £15M, depending on the year. Demand greatly exceeds supply and imports during the home season amount to another £15M. Grower interest in planting cherries and plums is high – stimulated by the development of improved cultivars, dwarfing rootstocks and cropping systems, and by demand from supermarkets. A effective vehicle for technology transfer exists in the form of the ‘Clubs’. The Cherry Club and the Plum Club were set up about ten years ago and subsequently merged to form the Stone Fruit Club. This was an excellent forum for interchange between research workers, growers, advisors and marketers via periodic meetings and newsletters. Moreover, it has taken the initiative in providing partial financial support for trials of new selections and varieties and in securing additional funds from the Horticultural Development Council (HDC). SID 5 (Rev. 3/06) Page 4 of 18 There are two important gaps in the portfolio of commercial varieties currently available, which lead to loss of market share and income. With cherries, the price per pound rises considerably during July as the imports from southern Europe fade away, but by the end of July the latest commercial varieties in the UK are themselves ripening and, thereafter, the supermarkets are supplied by high-priced imports, e.g. from Washington State, USA. Some new varieties ripen in early August, including ‘Penny’, which was released by East Malling Research (EMR) in 2001. There is certainly a need for varieties ripening from mid-August to September that meet the normal commercial requirements of, e.g., large fruit, black flesh, firm texture and good quality. At EMR we have breeding lines that are pickable until October, though these are small-fruited. For plum, the season starts with ‘Opal’ in early August and then there is a lull until ‘Victoria’ in late August. The varieties ‘Avalon’ and ‘Excalibur’, released from the Long Ashton programme, filled this gap and had excellent quality, but have been erratic in cropping and have an awkward upright tree habit. To maintain continuity of supply to the supermarkets, growers need an early mid-season variety, with good quality and reliable cropping and spreading habit. A second gap is in early September, between ‘Victoria’ and ‘Marjorie’s Seedling’. A prerequisite for reliable cropping in plum, which flowers early in the year when pollinating insects may be scarce, is self-compatibility. Another desirable feature is red or red and yellow fruit colour, which UK customers prefer to blue. However, in the case of cherry, it was also feasible to develop some interspecific breeding lines. These are important as they promise to be a source of novel useful characters that are not available in intraspecific breeding lines. We already have some diploid interspecific hybrids that appear to have resistances to cherry blackfly (Myzus cerasi) and cherry leaf spot (Coccomyces hiemalis), a fungal disease that has spread alarmingly in northern Europe in recent years. These characters, introgressed into sweet cherry, would allow a useful reduction in pesticide use. We have recently developed tetraploid forms of two firm-fleshed sweet cherry cultivars that should allow successful hybridisation with tetraploid species such as sour cherry, which have useful characteristics such as late ripening, small tree size and novel pest and disease resistances. This work is longer-term than the breeding for late ripening described earlier as two more generations of backcrossing to commercial cherries may be needed to improve fruit quality sufficiently. Moreover, in the case of cherry, it should be possible to increase the efficiency of the breeding programme by making some use of the molecular markers and maps recently developed at EMR. Traditional breeding comprises crossing, screening, selection and trialling and takes several years. The detection of markers genetically linked to useful characters paves the way for marker-assisted selection, whereby seedlings can be screened for the presence of the markers indicating desirable characters soon after germination. The project benefited from close links with the Defra projects on genetic improvement of top fruit project and of farm woodlands projects and, latterly, with the Defra project on rosaceous genomics. This work accorded with Defra’s policy for sustainable horticulture. The ROAME As for the horticulture AUs indicate support for work on breeding fruit crops for quality and for pest and disease resistance using conventional and genome-based techniques. The objectives are as follows: 1. To breed and select large-fruited, late-ripening cherry selections for grower-funded trial Promising selections identified for Stone Fruit Club trial Progenies raised for selection 2. To introgress pest and disease resistance from interspecific breeding lines into sweet cherry Hybrids, and perhaps backcrosses, screened for resistance to leaf spot and genetics clarified Resistant selections backcrossed to large-fruited selections to introgress resistance into sweet cherry 3. To raise interspecific progenies of sweet cherry at tetraploid level SID 5 (Rev. 3/06) Page 5 of 18 Sweet × sour cherry progenies raised for evaluation of vigour, blackfly resistance, ripening season and fruit quality 4. To identify and apply molecular markers to benefit of cherry improvement programme Microsatellite library of cherry developed and informative primers designed Marker for resistance to leaf spot identified to avoid need for resistance screening 5. To breed and identify large-fruited red plum selections ripening between ‘Opal’ and ‘Victoria’, or between ‘Victoria’ and ‘Marjorie’s Seedling’ for grower-funded trial. Promising individuals selected from existing seedlings passed to Stone Fruit Club for trial Progenies raised for selection of large-fruited red medium-early or medium-late plums 6. To transfer technology to the industry Late ripening cherry and medium-early or medium-late ripening plum selections propagated for Stone Fruit Club trials New cherry cultivars and selections in Stone Fruit Club trials genotyped for incompatibility alleles so appropriate pollinators can be recommended 7. To review progress of project Annual meetings held with Defra project officer Extent to which objectives were met The project successfully developed breeding lines and tools for the genetic improvement of cherries and plums for UK horticulture. The objectives were met in full, with the exception of objective 2. As explained in the text and in previous reports the tetraploid forms of sweet cherry were slow to flower; the sweet × sour crosses were made, but no seeds resulted at the first attempt Methods and Results Objective 1 ‘Penny’, a late-ripening black-fruited cultivar from the cross ‘Colney’ × ‘Inge’, which was released during the previous project, continued to perform very well on growers’ farms and demand for trees is high. Its only fault appears to be slow propagation; a high proportion of its buds are floral and unsuited for vegetative budding. Curent front runners in HDC-funded trials, and earmarked for release, are the EMR latish black selections C18-19 and C18-28 (‘Inge’ open pollinated), which have cropped very well. Selection The project inherited several thousand seedlings of various ages from intraspecific crosses made previously, largely with the objectives of late ripening and/or good fruit quality in mind. These had been planted out, when one or two years old, on their own roots on the seedling selection plots at 2.5 m × 4 m. When the trees had passed through their juvenile phase and were approximately six years old, they were netted, generally for one year only, against birds. No regular pruning was undertaken though trees were headed back as necessary to facilitate netting. Trees were often retained for a further year. The seedlings were observed and recorded in most years for, e.g., tree habit, field resistance to bacterial canker, flowering and initial fruit set. Netted trees were assessed for cropping, season, fruit colour, fruit size, texture, flavour and, in most years, resistance to rain-induced cracking. In two years, when bird damage was less severe than usual, fruit characters could also be assessed on the older unnetted material. Individuals deemed worthy of industry-funded trial – ideally with spreading habit and canker resistance and reliably bearing large, firm, well-flavoured late black fruit resistant to cracking – were budded or grafted on to the dwarfing rootstock Gisela 5 (G5) to provide about five replicates for trial. Additionally, several intraspecific progenies were recorded for the segregation of two apparently Mendelian characters, yellow-green leaf and tobacco-like leaf, with a view to clarifying inheritance patterns. SID 5 (Rev. 3/06) Page 6 of 18 In 2003, approximately 300 seedlings were netted against birds so that fruit could be assessed. Seven selections, C304-35 (‘Stark Hardy Giant’ × ‘Colney’), C308-4, -20, -64, -68 and OA (‘Colney’ × ‘Gaucher’) and C318-30 (‘Colney’ × ‘Stark Hardy Giant’), looked promising and were budded on to Gisela stocks in August 2003 for possible trial. In addition, it was possible to undertake some reassessment on older un-netted trees as bird damage was slow: seven more selections, C216-3 (C3-5 × ‘Lapins’), C247-1 (‘Colney’ × ‘Sunburst’), C250-1 and -2 (parentage uncertain), C261-52 (‘Merton Late’ × ‘Colney’), and C273-1 and -11 (‘Colney’ × ‘Sweet September’), had some merit and were likewise budded onto G5. (This material was subsequently planted in the HDC-funded variety trial MP183 in March 2005.) In 2004, approximately 450 intraspecific cherry seedlings were netted for detailed observation of fruit crop, quality and season. Seven selections were earmarked for for trial – they were C328-1 and -22 (‘Summit’ × ‘Strawberry Heart’), C338-3 and -14 (‘Stella’ × ‘Van’) and C358-1, -2 and -4 (‘Ferbolus’ × ‘Penny’) – and were propagated by bench grafting in March 2005 (This material was subsequently planted in HDC-funded variety trial MP187 in March 2006.) Also of interest was progeny C356 from the cross of ‘Colney’ × ‘Sweet September’; the seedlings were mostly late, with fruit retained into September, but fruit size was mediocre. In 2005, some 650 seedlings from intraspecific crosses were netted and four selections were provisionally short-listed. The seedlings were reassessed in 2006 – although the trees were unnetted, bird damage was less severe than usual – and the short-list was revised and expanded. Eventually, the list of six selections meriting trial from this block was finalised as: C336-10 (‘Lapins’ × ‘Van’), C3651 (‘Stark Hardy Giant’ × ‘Colney’), C367-9 (‘Ferbolus’ × ‘Colney’), C369-5 (‘Summit’ × ‘Colney’), C371-4 (‘Colney’ × ‘Sunburst’) and C374-5 (‘Colney’ × ‘Bing’). Of these, C367-9 was particularly interesting – resistant to rain-induced cracking in 2005 and fruit weighing up to 16 g. These selections were bench grafted in March 2007 and, along with the three mentioned in the next paragraph, are available for planting in March 2008. In 2006, the next batch of about 650 seedlings netted for assessment included some 350 seedlings of progeny C417 from the cross of ‘Colney’ × C210-7. Several of these were very late, with fruit still on the tree in early September, but then deteriorating rapidly, and several selections were of reasonable size. Two were short-listed for trial, C417-30 and -92, and C421-03 (‘Vic’ × ‘Colney’) was also shortlisted. These three were bench grafted in 2007 and, along with the six mentioned in the previous paragraph, are available for planting in March 2008. Subsequently C417-317 was thought worth trialling and graftwood was provided to an off-site propagator in March 2008. In 2007, about 500 trees were netted and, after evaluation, six selections were short-listed for trial. These were C445-17 (‘Sylvia’ × ‘Noir de Meched’), C454-2 (‘Sunburst’ × ‘Noir de Meched’), C458-1 (‘Penny’ × A53), C478-3 (‘Sasha’ × ‘Lapins’), C499-3 (‘Sweetheart’ × C273-18) and C512-6 (C18-19 × ‘Colney’). Graftwood of these, and of C417-317 as mentioned previously, was provided to an off-site propagator in March 2008. Genetic observations Several progenies from the cross ‘Donnissens Gelbe’ × ‘Alman Gulrod’, C295, C354, C381 and C507, 75 seedlings in all, and several progenies from the cross of ‘Donnissens Gelbe’ × ‘Victor’, C380, C418 and C508, 77 seedlings in all, were recorded for a transient yellow-green leaf phenotype, that is apparent in some seedlings for a week or two after budburst in cool weather. The character is presumably controlled by a recessive gene, though in both progenies the segregation ratios were 2:1 rather than 3:1. Minor inconsistencies in the segregation data occurred from one year to the next and so they need confirming before they can be used for mapping. Fifty-six seedlings from the backcross ‘Decumana’ (tobacco-like leaf) × C249-8 (normal leaf; ‘Charger’ × ‘Decumana’), belonging to progenies C403, C423, C463 and C506, were recorded for leaf phenotype. The segregation pattern of approximately 1:1 indicates a recessive gene for the tobaccolike leaf character. Crossing Controlled crosses were made in three years with the objectives indicated above, largely in an insectproof glasshouse using potted trees. To delay flowering of certain trees to be used as female parents SID 5 (Rev. 3/06) Page 7 of 18 until appropriate pollen was available, they were moved to the coldstore. In one year, severe frosts damaged flowers before pots were moved to the glasshouse. Generally the flowers of the female parents were emasculated for convenience of working, even though only a few cultivars are selfcompatible. It should be mentioned that in a few cultivars emasculation greatly reduced fruit set. Pollen viability of male parents was checked on agar plates; not infrequently the pollen collected at the beginning of the season was non-viable. If possible, about 1,000 flowers were pollinated per cross. If crosses were made on trees in the field, the pollinated flowers were bagged against insects. Fruit set was generally less than 50%. Fruit was harvested in the summer and the stones removed and cracked; in many batches fewer than 50% of seeds contained embryos. The embryos were sown in trays of compost and stratified in the coldstore for 16 weeks. Seedlings germinated in winter or early spring and were potted up; germination seldom exceeded 50% and often failed completely. Seedlings were planted out in the seedling selection plots a year or two later. In 2003, a frosty spring hampered crossing. Six good quality medium or late black cherries – ‘Penny’, ‘Regina’, ‘Sunburst’, ‘Noir de Meched’, ‘Nordwunder’ and ‘Tardif di Vignola’ – were pollinated with C273-18 (an exceptionally late ripening black cherry from the cross ‘Colney’ × ‘Sweet September’, but with fruit too small for commerce). However, only the first three crosses produced viable seed, which germinated in March 2004. The principal successful crosses made in 2004 were ‘Summer Sun’ (regular cropping) × C273-18, and ‘Summit’ (good quality black) × C210-7 (late ripening but smallfruited white; ‘Lapins’ × ‘Sweet September’). Successful crosses in 2005 were those of ‘Noir de Meched’, ‘Octavia’ and ‘Sasha’ (all black with excellent quality) × ‘Canada Giant’ (excellent quality and very large) and of ‘Summer Sun’ and ‘Summit’ × ‘Sunburst’ (self-compatible and very large). Material was collected from this last batch with a view to implementing marker-assisted selection for selfcompatibility and/or black flesh colour. Objective 2 Various interspecific progenies, numbering about 200 individuals, deriving from crosses of sweet cherry with species such as P. incisa and P. nipponica having resistance to black-fly (Myzus cerasi) – which causes stunting and severe twisting of the leaves – or leafspot (Coccomyces hiemalis) – which can cause premature defoliation – were already growing in the field at the start of the project. In 2003 seedlings of three families, C307 (‘Napoleon’ × C61-38 (‘Napoleon’ × P. nipponica)), C301 (‘Napoleon’ × C37-11 (‘Napoleon’ × P. incisa)) and C321 (C37-11 × C61-38), were netted and assessed for fertility, fruit characters and field resistance to pests and diseases. In addition records of older unnetted progenies were reviewed. Likewise in 2004, 2005, 2006 and 2007 two, five, three and three interspecific progenies from backcrosses of P. avium × P. incisa selections to P. avium, were netted and assessed. Fertility was generally poor but occasional seedlings cropped well. And, as expected, fruit size was larger than that of the original species. Some 20 hybrids were propagated on to Gisela to raise plants for controlled inoculation tests for resistance and/or for possible use as parents to introgress useful characters. Approximately 40 selections from the interspecific cross ‘Napoleon’ (susceptible to leaf spot) × P. nipponica (resistant) had been sent as graftwood in February to BAZ Dresden-Pillnitz as part of a collaboration funded by The British Council. In summer 2003 these were tested in a laboratory assay there by our collaborator Mirko Schuster for resistance to leaf spot using detached leaves. And in summer 2004 they were scored there for resistance to leaf spot on the basis of symptoms in the field. The agreement between the two data sets was not very strong but, when they were combined, 10 seedlings were provisionally classed as susceptible. However, if P. nipponica has a dominant gene for resistance, a segregation ratio of 1 resistant:1 susceptible rather than 3 resistant:1 susceptible would have been expected. The original CSG7 had a milestone to map the putative resistance gene; however, in the absence of robust phenotypic data, and indeed of evidence for a major resistance gene, this milestone was replaced. In 2007, some 20 interspecific selections from the progenies C301 (‘Napoleon’ × C37-11), C311 (‘Napoleon’ × C57-46), and C317, C361 and C372 (all ‘Colney’ × C57-38), with three grafted plants per selection and various parents as controls, were tested for resistance to blackfly in the glasshouse. The plants were growing in pots, tied to a cane and in compost free of the insecticide Intercept which is usually incorporated to suppress vine-weevil. In late May, five aphids were transferred to each shoot tip and colonization and leaf twisting were recorded over six weeks. All the C301s were susceptible but C311-3, C317-1, C361-3 and -7 and, particularly, C372-1 were as resistant as their resistant parent. SID 5 (Rev. 3/06) Page 8 of 18 On the basis of this small test, C57-38 and perhaps C57-46 are more effective donors of resistance than C37-11. It should be borne in mind that this was a test of resistance to the multiplication of the aphids; in the field it is possible that resistance results from plants not attracting egg-laying aphids in the autumn. In three years, further crosses were made under glass using some of the more fertile hybrids resistant to blackfly using the methods indicated in objective 1 with the aim of introgressing this resistance into P. avium. In 2003 the resistant interspecific hybrids C57-6, C57-43 and C57-46 (‘Napoleon’ × P. incise) were crossed in one direction or the other with the good quality cherries ‘Sasha’, ‘Summersun’, ‘Sunburst’, ‘Van’, A43 and 9239-1, ~1200 flowers in all. Only the crosses ‘Sasha’ × C57-6 and ‘Sunburst’ × C57-46, produced viable seed. In 2004, C57-46 (‘Napoleon’ × P. incisa) was backcrossed on to ‘Summersun’ (400 flowers giving 115 fruit) In 2006 crosses made included ‘Penny’, ‘Octavia’ and ‘Regina’, all of excellent quality, × C57-46 or × C37-17 (Napoleon × P. incisa). From 700 flowers pollinated, 30 seeds were produced and sown. The new wave of interspecific hybrids has yet to flower. In 2007, about 20 unreplicated seedlings raised from the crosses made in 2006 of ‘Octavia’ and ‘Penny’ × C57-46 were tested in the glasshouse for resistance to blackfly, as just indicated for the selections. Unfortunately, the seedlings, which had germinated in the winter, stopped growing in late May and the test, which requires young leaves, was inconclusive. Objective 3 Young, potted ex-micropropagation plants of induced tetraploids of the sweet cherries ‘Stella’, clone 79, and ‘Summit’, clone 130, were available at the beginning of the project but were expected to take several years to flower on their own roots. In 2004 they were bench grafted on to Colt rootstocks, not the most precocious stock, but one known to show generally good compatibility. Two grafted plants of each of the tetraploid clones on ‘Colt’ survived and grew well, with characteristically large, broad leaves. In 2005 each clone was grafted on to the dwarf rootstock G5 to raise additional trees, in the expectation that these might flower sooner for intercrossing with sour cherry. The tetraploid forms grafted on Colt in 2004 and on G5 in 2005 grew well in 2005 but did not initiate flower buds for 2006. In spring 2007 there were a few flowers on both sets of trees, but the trees on G5 were unhealthy. The small amount of available pollen was used to pollinate the large-fruited, naturally tetraploid sour cherry ‘Koros’ in the hope of creating fertile tetraploid interspecific hybrids. However, no fruit was set on that occasion. An additional strategy to produce tetraploid (i.e. fertile) hybrids of sour × sweet cherry was adopted in 2006 – relying on the “stylar sieving” effect whereby only occasional heteroallelic diploid pollen of a sweet cherry could succeed on the sour cherry style while normal haploid pollen would be rejected. The tetraploid sour cherry cultivar ‘Ujfehertoi Furtos’, having the incompatibility genotype S1S4SBSD was pollinated by the diploid sweet cherry ‘Sylvia’, S1S4. Nominally this cross is incompatible as haploid S1 or S4 pollen will fail. However, if rare diploid pollen of genotype S1S4 is produced, it should be compatible because of the phenomenon of ’competitive interaction’ (in which the alleles effectively cancel each other out). From 300 flowers pollinated, three seeds were produced and sown, but failed to germinate. Objective 4 Microsatellites To develop microsatellite markers of cherry origin for use in fingerprinting and mapping, a genomic library enriched for simple sequence repeat sequences was prepared from DNA of ‘Napoleon’. Clones were sequenced and primers were designed for sequences flanking the microsatellites. Primers were screened on a set of 14 sweet cherry cultivars to identify those revealing polymorphism. A primer note describing 21 such primers, EMPA1 to 21, was published in Molecular Ecology Notes in 2004. The primers are among those used to construct a genetic map as described in the next section. Mapping The microsatellite map based on a progeny from the cross of Napoleon × Prunus nipponica that was initiated before this project to extend the existing isoenzyme map was completed for publication. An additional 44 seedlings were propagated on Colt rootstock to add to the mapping progeny of 50 that SID 5 (Rev. 3/06) Page 9 of 18 had been used for the original isoenzyme mapping so that the resolution of the map could be improved, and DNA extracts of all 94 seedlings were prepared. In all, 260 primers developed in various Prunus species, but especially peach and cherry, were used to amplify the DNAs from the parents and from a set of seedlings by PCR and checked for segregation (180 in this project and 80 in preliminary work). The 160 microsatellites that segregated were then scored in the full progeny, using fluorescent primers and automated sizing on an ABI prism sequencer. In addition, in collaboration with the rosaceous genomics project HH3724SSF, five known function genes and the incompatibility locus S were included. The cosegregation data from the markers were used to construct a linkage map with JOINMAP software of about 170 loci for cherry which spanned 680 cM over eight linkage groups with an average marker spacing of 3.9 cM/marker and just five gaps longer than 15 cM. Markers previously mapped in P. dulcis ‘Texas’ × P. persica ‘Earlygold’ allowed the cherry map to be anchored to the peach × almond map and showed the high level of synteny between the species. Eighty-four loci segregated in P. avium ‘Napoleon’ versus 159 in P. nipponica and so the level of heterozygosity in ‘Napoleon’ with respect to markers scored was not high enough for good coverage of the sweet cherry genome. In addition, 71 isoenzyme loci scored on a subset of 47 seedlings (39 in previous work and 32 more recently) have been included in the map. The microsatellite loci are conveniently transferable to progenies in which segregating agronomic traits can be mapped and the isoenzyme loci and known function genes are ideal for comparisons of synteny with other genera such as Fragaria (strawberry) and Malus (apple). A paper reporting the map was submitted to Tree Genetics and Genomics in 2007. Previous work had detected an AFLP marker for the Bf gene for black flesh colour in a small segregating progeny of 45 seedlings from the cross ‘Bradbourne Black’ (black) × ‘Merton Late’ (white). In collaboration with the rosaceous genomics project HH3724SSF, DNA extracts from the seedlings were amplified with a set of 20 microsatellite markers known from the ‘Napoleon’ × P. nipponica map to be well distributed across the Prunus genome. In this way the Bf gene was located on linkage group 3. Then a larger progeny of 75 seedlings from the cross ‘Napoleon’ (white) × ‘Merton Heart’ (black) which had already been scored for flesh colour was analysed for a set of 10 microsatellite markers known to map to linkage group 3. By analysing co-segregations, a map locating Bf very close to AMPA119 on linkage group 3 was produced. When a set of cultivars was analysed for AMPA119, it proved to be frequently heterozygous, indicating its use as a marker for pre-selection of black fleshed seedlings, provided the linkage ‘phase’ (coupling or repulsion) in the black-fleshed parent is known. A paper reporting this work has been prepared. In addition, the locus for yellow skin colour ys, a recessive character segregating approximately 1:1 in the progeny of ‘Donnissens Gelbe’ (yellow skin) × ‘Alman Gulrod’ (blush) mentioned earlier, was mapped and located on linkage group 3. Incompatibility Papers describing the use of fluorescent consensus primers amplifying across the first intron of the SRNase gene or across the intron of the SFB gene to determine S genotype in cherries were published in Plant Breeding and TAG, respectively, in 2006. Work to understand the nature of the self-compatible alleles S3’ and S4’, which are of considerable agronomic importance as self-compatible cultivars can be grown without pollinators, was completed in collaboration with the University of Nottingham and the Defra project on diversity of broad-leaved trees. Sequencing of the SFB genes of these pollen-part mutants showed that the first is due to a deletion of the SFB gene and the second to a premature stop codon causing the SFB protein to be truncated. A landmark paper describing this work was published in The Plant Cell in 2005. With supplementary funding from the University of Palermo, a novel form of self-compatibility in the Sicilian sweet cherry cultivar ‘Kronio’ was characterised. Crossing tests and sequencing of the SRNase and SFB alleles indicate the self-compatibility is due to a natural pollen-part mutant of allele S5, denoted S5': a mutation to a premature stop codon causes the SFB protein to be truncated. Previously only induced pollen-part mutations have been reported in cherry. An allele-specific primer for an associated microsatellite in the S-RNase second intron was developed which can be used for markerassisted selection as the length of the microsatellite differs between the S5 (self-incompatible) and S5' (self-compatible) versions. An account of this work was published in Journal of Experimental Botany in 2007. SID 5 (Rev. 3/06) Page 10 of 18 Objective 5 The project inherited some 300 plum seedlings, mostly from crosses made in 1997 and 1998, growing in the seedling selection plots on their own roots and receiving minimal pruning. Only about 5% of them cropped in 2003 and 30% in 2004 and it was not until 2005 that most cropped and could be evaluated for season, yield, fruit colour, size and eating quality. The seedling juvenile period seems to be rather longer than that of cherry. In 2004, seedling P6-19 (‘Opal’ × ‘Herman’) was interesting; the fruit was early, round, medium-large, blue, sweet and free-stone and the tree was not too twiggy. In 2005 P6-19 again looked good, as did P2-2 and -18 (WJ27 × ‘Warwickshire Drooper’) and P4-9 (‘Avalon’ × ‘Warwickshire Drooper’). These three were short-listed for industry-funded trial, and bench-grafted in March 2006. (However the resulting trees planted in trial in March 2007 failed to establish.) In 2006 P7-38 (‘Opal’ × ‘Avalon’) was outstanding, with a heavy crop of large blue free-stone fruit ripening before ‘Opal’, though with rather upright and twiggy growth habit, and was earmarked for trial. In March 2007, P6-19 and P7-38 were bench-grafted on St Julien A (and, at the time of writing, trees are available for planting in trial). A PhD student who had been working at EMR on the molecular genetics of incompatibility (S) in diploid and hexaploid plum submitted his successful thesis in 2004. A refereed paper from this work was published in Plant Breeding in 2004, reporting a pair of primers that seem particularly effective at amplifying the polymorphic second introns in plum S alleles to facilitate genotyping and exploring the genotypes determining self-compatibility. In 2008, a paper was published in Molecular Genetics and Genomics, which draws attention to the near sequence identity of the coding regions, but not of the introns, of various alleles in plum with those in other stone-fruit, examples of trans-specific polymorphism. Some 14 plum crosses were attempted in the glasshouse in 2005, with up to 500 flowers pollinated per cross. The most successful involved the parents ‘Avalon’ and ‘Excalibur’ (excellent quality, red colour), ‘Jojo’ (resistant to plum pox, self-fertile), ‘Jubileum’ (good quality,self-fertile), ‘Opal’ (good quality, early), ‘Reine Claude de Bavay’ (excellent quality, self-fertile), ‘Victoria’ (self-fertile, good quality, red colour) and ‘Voyager’ (good quality, self-fertile). About 300 embryos were sown and 75 seedlings germinated and were potted up. An additional batch of plum crosses was made in 2006, of ‘Anita’ (exceptionally large fruit) and ‘Jojo’ with cultivars of good fruit quality, including gages. Some 230 embryos were sown and 130 seedlings germinated and were potted up. Objective 6 Several selections from the forerunner project performed well in industry-funded trial. Thus, according to the Horticultural Development Council’s 2005 report, “The new EMR selections C3-30, C17-5, C1819 and C18-28 have impressed with their exceptional cropping on young trees and their low susceptibility to cracking and rotting”. In 2006, both C18-19 and C18-28 (‘Inge’ open pollinated) looked impressive in trial MP169, planted March 2001, and received favourable comment at the well-attended Cherry Walk. These two are recommended for release. Younger selections looking promising in trial include C216-3 (C3-5 × ‘Lapins’) in MP183, planted March 2005, and C269-8 (‘Van’ × ‘Colney’) and C318-11 (‘Colney’ × ‘Stark Hardy Giant’) in MP179, planted March 2004. In March 2004, trees on G5 rootstocks of five selections, C269-8 (‘Van’ × ‘Colney’), C304-35 (‘Stark Hardy Giant’ × ‘Colney’, C316-05 (‘Sylvia’ × ‘Hertford’), and C318-11 and -38 (‘Colney’ × ‘Stark Hardy Giant’), that were identified as promising in the forerunner project, were provided for planting in trial MP179 funded by the HDC and the Stone Fruit Club. In March 2005 and 2006, trees on G5 of 14 cherry selections (C216-3, C247-1, C250-1 and -2, C261-52, C273-1 and C273-11, C304-35, C308-4, C308-20, C308-64, C308-68 and OA, and C318-30) and seven cherry selections (C328-1 and C32822, C338-3 and C338-14 and C358-1, C358-2 and C358-4) respectively, as detailed in objective 1, were planted in industry funded trials MP183 and MP187. In addition, nine cherry selections (C336-10, C365-1, C367-9, C369-5, C371-4, C374-5, C417-30 and C417-92 and C421-3) and two plum selections (P6-19 and P7-38) as detailed above are available for planting in industry-funded trial in March 2008; and trees of the seven cherry selections just passed to an off-site propagator (C417-317, C445-17, C454-2, C458-1, C478-3, C499-3 and C512-6) should be available for planting in March 2009. SID 5 (Rev. 3/06) Page 11 of 18 In addition to the trials of scion selections, four cherry rootstock selections, all derived from P. mugus in the forerunner project, namely C113-3, C376-1, C376-4 and C376-5, worked to ‘Sunburst’ were planted in industry funded trial MP182 in March 2005 to assess their dwarfing effect and orchard performance. An account of the cherry breeding programme, and an exhibit of selections being considered for trial by the Stone Fruit Club, was presented at the East Malling Research Association (EMRA) Members’ Day in August 2003. In addition, a compilation of incompatibility genotypes of ~200 cultivars, determined at EMR with protein or DNA techniques and elsewhere, and including all those in Stone Fruit Club trials, was presented (and subsequently published). The breeding work was well received and two very supportive articles subsequently appeared in The Fruit Grower. A presentation about the plum breeding programme was made at a well-attended Plum Walk, organised for the East Malling Research Association and the Stone Fruit Club, in early August 2005 and medium-early plum seedlings being considered for trial were shown to growers and advisors present; the opportunity was also taken to display some promising medium-late cherry scion selections. Again there was encouraging coverage in The Fruit Grower. Three outstanding cherry seedling selections were exhibited at the Stone Fruit Club Cherry Walk in July 2006, at which participants were able to inspect the selections in trial. To determine the incompatibility groups of some of the EMR cherry selections in industry-funded trial, DNA was extracted from leaves of 15 selections – five planted in 1998 (MP162) and 10 planted in 2001 (MP169) – and amplified with primers flanking the introns of the S-RNase gene; the products were separated on agarose. By comparison with reference cultivars of known genotype all 15 selections were genotyped and assigned to pollination groups (Table 1). This information has been provided to staff conducting these trials for inclusion in the next HDC fact sheet. Two of the selections had an apparently new allele which, for the time being, has been labelled Sx. In addition, with inputs from visiting workers funded by the Mount Trust and by British Council, the 200 or so sweet cherries in Defra’s National Cherry Collection at Brogdale were genotyped at the S locus. DNA was extracted and amplified using two fluorescently labelled primer pairs developed at EMR, one amplifying across the first intron of the S-RNase gene and the other amplifying across the intron associated with the SFB gene. The amplification products were sized automatically on an ABI sequencer – which gave far more precise results than are available from separation on agarose gels. As well as providing useful information regarding pollinators for a forthcoming catalogue of the collection, this study also indicated various sets of possible synonyms. Subsequently, with inputs from a visiting worker funded by the University of Palermo, the sweet cherry accessions from Brogdale were fingerprinted with 13 microsatellites, that could be analysed conveniently in three multiplexes. Approximately one quarter proved to be duplicates! Subsequently Defra commissioned EMR to fingerprint the Brogdale apple and pear collections. Table 1. Incompatibility genotypes of cherry selections in Stone Fruit Club trial plantings S genotype Pollination group Trial MP162 (planted 1998) C11-18 C73-5 (= Penny) C90-24 C90-30 C105-3 S5Sx S6S9 S3S6 S3S5 S1S5 New X VI VII XIV Trial MP169 (planted 2001) C3-27 C3-28 C3-30 C3-44 C12-14 C12-17 C17-5 C18-19 C18-28 S5S9 S1S4 S3S9 S3S9 S5Sx S1S6 S3S4 S1S9 S6S9 XXXVII IX XVI XVI New XX III XVIII X SID 5 (Rev. 3/06) Page 12 of 18 Several seedlings from crosses made to investigate the nature of self-compatibility in the John Innes selection JI14137, a derivative of JI2538, were provided to a BBSRC-funded project conducted by Dr Tineke Sonneveld at the University of Nottingham. The primers for the incompatibility locus developed during this project have also been widely adopted for other Prunus crops and used by visiting workers at EMR. At the ECPGR Fruit Network Workshop on Microsatellite Fingerprinting run by EMR in December 2006, EMR’s recommendations for a set of 16 microsatellite markers and general guidelines for use in fingerprinting cherry collections internationally, to facilitate the comparison of data sets, was adopted. In addition, technology was transferred via an extensive series of publications and talks as detailed below in Actions Arising and a large number of research papers as detailed in section 9. And advice was given to growers over the telephone on such matters as choice of cultivar and pollination. Objective 7 Project progress and the merits of several cherry and plum selections was discussed with the project officer, Emma Hennessey, and her colleague, Robert Bradburne, at East Malling on 28 July 2004. An additional meeting took place in July 2006 and some points were discussed by email. Progress on the project was presented at the Defra Reviews in London in September 2005. A positive report was received later and was discussed with Defra staff. Discussion and Implications This project has been very effective at generating genetic information – especially relating to maps, markers and incompatibility – and promising selections – especially of late cherry – for the genetic improvement of the UK’s stone-fruit crops. Thus, the map of 160 microsatellite markers derived from ‘Napoleon’ × P. nipponica is the best cherry map available, due to the high proportion of segregating loci. It proved its use in providing a framework for the mapping of the loci for black flesh and yellow skin colour, and would be useful for mapping the yellow-green leaf and tobacco-like leaf traits. The marker for black flesh allows marker-assisted selection for this agronomically important trait. Supplemented with known function genes, the ‘Napoleon’ × P. nipponica map would provide an ideal framework for studies of synteny with Malus and Fragaria. The work on incompatibility has not only developed innovative primers for detecting alleles and genotyping cultivars, selections and seedlings but has clarified the molecular genetics of three selfcompatible mutants. Regarding breeding lines, 42 promising cherries and two promising plums were planted in, and/or propagated for, industry-funded trial during the life of the project and several cultivars are likely to be released in the next six years. And several younger seedlings, currently in the field or yet to be planted out, are likely to merit trial. The programme of technology transfer – including material, articles, presentations, research papers and provision of advice – has been extensive. The project benefited greatly from close interaction with the complementary Defra projects on rosaceous genomics, and also from links with the Stone Fruit Club. In addition, supplementary funding from the Mount Trust, the University of Palermo and The British Council created enormous ‘added value’. It is the responsibility of The Brogdale Advisory Committee to consider if some of the genetic resources are maintained at Brogdale. Of particular importance are the ‘Napoleon’ × P. nipponica mapping progeny, various cultivars not already in the national collection, a set of Prunus species accessions including donors of pest and disease resistance, and various cherries and plums of genetic interest, especially the reference accessions for particular (in)compatibility alleles. SID 5 (Rev. 3/06) Page 13 of 18 Possible future work Regarding breeding lines there are opportunities for identifying further selections for trial and indeed for making further crosses among those entered for trial or possessing pest or disease resistance. Ideally, targeted surveys should be conducted, e.g. to detect late-ripening types among the wild cherries collected by the Defra-funded project on the genetic improvement of farm woodland species and to detect additional resistances among accessions of wild Prunus species. Moreover, with climate change a serious concern, attention could be paid to screening for water use efficiency and winterchilling requirement with a view to breeding for these characters. Regarding marker work, there is the opportunity to map a few more single gene characters that have recently been detected and, in due course, and with appropriate progenies, to map components of, e.g., late ripening and winter chilling requirement. To use these markers efficiently it may be possible to design multiplexes. To facilitate the characterisation of agronomic genes, a resource such as a BAC library is needed to bridge the gap between marker identification and gene isolation. It is likely that establishing regions of synteny among the various rosaceous genera will facilitate the transfer of marker information across crops and the identification of genes for homologous traits. In addition to their direct use in genetic improvement, the microsatellites and markers for the incompatibility locus have opened up opportunities for understanding population genetics in wild material, elucidating patterns of domestication and producing standardised fingerprints of germplasm collections in UK and elsewhere. Indeed these markers are often transferable to other Prunus species. Actions arising including IP, presentations and publications Intellectual Property Several EMR cherry selections are already in industry-funded trials and, as reported, the heavy cropping black-fruited selections C18-19 and C18-28 appear to be worth releasing as cultivars. EMR will seek EU Plant Breeders’ Rights when appropriate. As described in the main text, 42 cherry selections and two plum selections have been planted in or propagated for industry-funded trial during this project. Thus it is expected that further cultivars will be released in due course. Non-refereed publications Tobutt, K.R., 2003. Cherry incompatibility groups. EMRA Members Day Report – August 2003, pp 811. Tobutt, K.R., 2003. Cherry breeding. EMRA Members Day Report – August 2003, pp 29-30. Bekefi, Z., Tobutt, K.R., & Sonneveld, T., 2004. Genotyping Hungarian sweet cherry cultivars for selfincompatibility. Acta Horticulturae 663:657-660. Clarke, J.B., Ortega, E., Sutherland, B., Marchese, A. & Tobutt, K.R., 2004. Some new cherry microsatellites and their transferability to other stone fruits. Acta Horticulturae 663:83-86. Schuster, M., & Tobutt, K.R., 2004. Screening of cherries for resistance to leaf spot, Blumeriella jaapii. Acta Horticulturae 663:239-243. Sutherland, B.G., Tobutt, K.R., & Robbins, T.P., 2004. Molecular genetics of self-incompatibility in plums. Acta Horticulturae 663:557-562 Tobutt, K.R., 2004. Extending the season of stone fruit by breeding. Plant it! Defra R&D Newsletter 6:7. Tobutt, K.R., Sonneveld, T., Bekefi, Z., & Boskovic, R., 2004. Cherry (in)compatibility genotypes – an updated cultivar table. Acta Horticulturae 663:667-671. Sutherland, B.G, Tobutt, K.R., & Robbins, T.P., 2007. Molecular genotyping of self-incompatible plum cultivars. Acta Horticulturae 734:47-51. SID 5 (Rev. 3/06) Page 14 of 18 Clarke, J.B., Tobutt, K.R. in press. A standard set of accessions, microsatellites and genotypes for harmonising the fingerprinting of cherry collections for the ECPGR. Eucarpia 2007 Acta Horticulturae (submitted). Presentations Tobutt, K.R. ‘Molecular markers in Prunus – some observations’. Presentation to ECP/GR Prunus Working Group, Budapest, 20 June 2003. Tobutt, K.R. ‘Cherry incompatibility groups’. Talk and poster at EMRA Members Day, East Malling, 7 August 2003 Tobutt, K.R. ‘Cherry breeding’. Talk on plots at EMRA Members Day, East Malling, 7 August 2003. Tobutt, K.R. ‘Breeding fruit, broadleaved timber trees and woody ornamentals’. Talk at glasshouses to visitors to EMR launch, East Malling, 17 March 2004. Tobutt, K.R. ‘Cherry breeding at East Malling’. Talk to Royal Horticultural Society Fruit Group, RHS Gardens, Wisley, 22 March 2004. Zanetto, A., le Dantec, L., Renoux, T., Dosba, F., Maggioni, L., Tobutt, K.R. ‘The Prunus European Cooperative Programme for Genetic Resources – a networking activity’. Poster at Eucarpia Symposium, Angers, September 2003. Tobutt, K.R., Sonneveld, T., Bekefi, Z. ‘Cherry (in)compatibility genotypes – an updated cultivar table’. Poster at Eucarpia Symposium, Angers, September 2003. Schuster, M., Tobutt, K.R. ‘Screening of cherries for resistance to leafspot, Blumeriella jaapii’. Poster at Eucarpia Symposium, Angers, September 2003. Clarke, J.B., Ortega, E., Sutherland, B., Marchese A., Tobutt, K.R. ‘Some new cherry microsatellites and their transferability to other stone fruits. Poster at Eucarpia Symposium, Angers, September 2003. Sutherland, B.G. ‘Incompatibility studies in plums’. September 2003. Presentation to Eucarpia Symposium, Angers, Sutherland, B.G. Molecular genotyping of self-incompatible plum cultivars. Lecture to ISHS Symposium on Plum and Prune, Ullensvang, Norway, September 2004. Tobutt, K.R. ‘Cherry breeding at East Malling Research’. Lecture to Northern Fruit Group, Harlow Carr, 21 May 2005. Tobutt, K.R. ‘Cherry breeding at East Malling’. Lecture to Friends of Brogdale, Brogdale, 17 July 2005. Tobutt, K R. ‘Plum breeding at East Malling Research’. Presentation at EMRA Plum Walk, East Malling, 4 August 2005. Tobutt, K.R. ‘Late cherries [and HNS]’. Talk to Kent Nursery Stock Group, East Malling, 31 August 2005. Tobutt, K.R. ‘Extending the season of stone fruit by breeding late-ripening cherries and medium-early and medium-late plums’. Presentation at Defra Project Reviews, London, 21 September 2005. Tobutt, K.R. ‘Microsatellite markers for managing Prunus collections’. Presentation to ECP/GR Prunus Working Group, Larnaca, 2 December 2005. Tobutt, K.R., Govan, C, Evans, K.M., Spencer, J.E. ‘Top fruit selections in the pipeline’. EMR Trade Open Day, 22 September 2005. Tobutt, K.R. ‘Breeding cherries at East Malling Research’. Poster presentation at National Fruit Show, Detling, 13 October 2005. SID 5 (Rev. 3/06) Page 15 of 18 Tobutt, K.R. ‘Aspects of perennial plant breeding relevant to the A-level syllabus’. Talk to pupils of Sutton Valence School, East Malling Research, 21 February 2006. Tobutt, K.R. ‘Molecular markers for fingerprinting and marker-assisted selection in fruit’. Talk to RHS Fruit and Vegetable Committee, Vincent Square, London, 11 April 2006. Tobutt, K.R. ‘Cherry breeding and varieties’. Talk to West Sussex Fruit Group, East Malling, 18 July 2006. Tobutt, K.R. ‘Three promising cherry selections from EMR’. Presentation at Stone Fruit Club Cherry Walk, East Malling, 18 July 2006. Tobutt, K.R. ‘Cherry breeding’. Talk to RHS Fruit Group, East Malling, 5 September 2006. Tobutt, K.R. ‘Introduction to ECPGR Fruit Network Microsatellite Workshop’. Presentation at ECPGR Microsatellite Workshop, East Malling, 7 December 2006. Tobutt, K.R. ‘Microsatellites for fingerprinting Prunus for ECPGR Database’. Presentation at ECPGR Microsatellite Workshop, East Malling, 7 December 2006. Tobutt, K.R. ‘Stone fruit breeding at EMR and some promising selections’. Talk to Brenchley & East Sussex Top Fruit Discussion Group, Marden, 19 April 2007. Tobutt, K.R. ‘Cherry improvement at EMR’. Seminar to staff, East Malling, 15 May 2007. Clarke, J.B., Tobutt, K.R. A standard set of accessions, microsatellites and genotypes for harmonising the fingerprinting of cherry collections for the ECPGR. Poster at Eucarpia Symposium, Zaragoza, September 2007. Clarke, J.B., Sargent, D.J., Belaj, A., Tobutt, K.R. Sequence-tagged site (STS) cherry map. Poster at Rosaceous Genomics Meeting, Aylesford Friary, 6 December 2007 Tobutt, K.R., Boskovic, R.I. ‘Self-(in)compatibility of diploid and polyploid Prunus’ – a review of work at EMR. Presentation at Rosaceous Genomics Meeting, Aylesford Friary, 6 December 2007. Knowledge transfer An account of various other aspects of knowledge transfer from this project is given above under objective 6 and refereed publications are presented below in section 9. 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. SID 5 (Rev. 3/06) Page 16 of 18 Bekefi, Z., Tobutt, K.R. & Sonneveld, T., 2003. Determination of (in)compatibility genotypes of Hungarian sweet cherry (Prunus avium L.) accessions by PCR-based methods. International Journal of Horticultural Science, 9(3-4):37-42. Clarke, J.B. & Tobutt, K.R., 2003. Development and characterisation of polymorphic microsatellites from Prunus avium ‘Napoleon’. Molecular Ecology Notes 3:578-580. Sonneveld, T,. Tobutt, K.R. & Robbins, T.P., 2003. Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers. Theoretical & Applied Genetics 107:1059-1070. Sutherland, B.G., Robbins, T.P., & Tobutt, K.R., 2004. Primers amplifying a range of Prunus Salleles. Molecular Ecology Notes 123:582-584. Tobutt, K.R., Bošković, R., Cerović, R., Sonneveld & T., Ruzić, D., 2004. Identification of incompatibility alleles in the tetraploid species sour cherry. Theoretical & Applied Genetics 108:775-785. De Cuyper, B., Sonneveld, T. & Tobutt, K.R. 2005. Determining self-incompatibility genotypes in Belgian wild cherries. Molecular Ecology 14:945-955. Marchese, A., Tobutt, K.R. & Caruso, T., 2005. Molecular characterisation of Sicilian Prunus persica cultivars using microsatellites. Journal of Horticultural Science & Biotechnology 80:121-129. Ortega, E,. Sutherland, B.G., Dicenta, F., Bošković, R. & Tobutt, K.R. 2005. Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes. Plant Breeding 124:188-196. Sonneveld, T, Tobutt, K.R, Vaughan, S.P. & Robbins, T.P., 2005. Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-Box gene. The Plant Cell 17:37-51. Boskovic, R.I, Wolfram, B, Tobutt, K.R., Cerovic, R., Sonneveld T. (2006) Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry. Theoretical and Applied Genetics 112:315-326. Sonneveld, T., Robbins, T.P., Tobutt, K.R. (2006) Improved discrimination of self-incompatibility SRNase alleles in cherry and high throughput genotyping by automated sizing of first intron polymerase chain reaction products. Plant Breeding 125:1-3. Ortega, E., Bošković, R.I., Sargent, D.J. & Tobutt, K.R. (2006). Analysis of S-RNase alleles of almond (Prunus dulcis): characterization of new sequences, resolution of synonyms and evidence of intragenic recombination. Molecular and General Genomics 276:413-426. Vaughan, S. P., Russell, K., Sargent, D.J., Tobutt, K.R. (2006). Isolation of S locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype. Theoretical and Applied Genetics 112:856-866. Bošković, R.I.,. Tobutt, K.R., Ortega, E., Sutherland, B., Godini, A. (2007) Self-(in)compatibility of the almonds P. dulcis and P. webbii: detection and cloning of ‘wild-type Sf’ and new selfcompatibility alleles encoding inactive S-RNases. Molecular and General Genomics 278:665676. Marchese, A., Tobutt, K.R., Raimondo, A., Motisi, A., Bošković, R.I., Clarke, J. & Caruso, T. (2007). Morphological characteristics, microsatellite fingerprinting and determination of incompatibility genotypes of Sicilian sweet cherry cultivars. Journal of Horticultural Science & Biotechnology 82:41-48. Marchese, A., Bošković, R.I., Caruso, T., Raimondo, A., Cutuli, M., Tobutt, K.R. (2007) A new selfcompatibility haplotype in the sweet cherry ‘Kronio’, S5′, attributable to a pollen-part mutation in the SFB gene. Journal of Experimental Botany 58:4347-4356. Šurbanovski, N., Tobutt, K.R., Konstantinović, M., Maksimović, V., Sargent, D.J., Stevanović, V., Ortega, E., & Bošković, R.I. (2007). Self-incompatibility of Prunus tenella and evidence that reproductively isolated species of Prunus have different SFB alleles coupled with an identical S-RNase allele. The Plant Journal 50:723-734. Marchese, A., Bošković, R.I.,. Martínez-García P.J., Tobutt, K.R. (2008) The origin of the selfcompatible almond ‘Supernova’. Plant Breeding 127:105-107. Sutherland B.G., Tobutt K.R., Robbins T.P. (2008) Trans-specific S-RNase and SFB alleles in Prunus self-incompatibility haplotypes. Molecular Genetics and Genomics 279:95-106. Vaughan, S.P., Bošković, R.I., Gisbert-Climent, A., Russell, K., Tobutt, K.R. (2008) Characterisation of novel S-alleles from cherry (Prunus avium L.). Tree Genetics & Genomics (accepted) Clarke, J.B., Sargent, D.J., Bošković, R.I., Belaj, A., Tobutt, K.R. (submitted) A cherry map from the interspecific cross Prunus avium ‘Napoleon’ × P. nipponica based on microsatellite, genespecific and isoenzyme markers. 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