Eneral enquiries on this form should be made to

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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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

We agree for the main report to be published on the website but would wish to withhold the Appendix showing the map of M432. We are planning to publish the map and do not wish this to be jeopardised.

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.

Marker-assisted selection (MAS) involves identifying genetic variation, typically in protein or DNA sequences that can be detected readily in the laboratory that is linked to a specific phenotype. This is done by correlating the presence of the genetic variant with the phenotype in a segregating progeny. Subsequently, seedlings can be screened soon after germination for the presence of the markers indicating desirable characters, thus increasing the efficiency of the selection process within a breeding programme. Microsatellite markers were the marker of choice for this project as they have the advantages of being co-dominant and highly variable – so they are readily transferable from one progeny to another – and detectable on an automated sequencer after the convenient laboratory technique of the polymerase chain reaction (PCR). This project capitalises on our current genetic expertise (including the new Defra/EU HiDRAS ‘High-quality disease resistant apples for a sustainable agriculture’ project) and breeding lines to increase the efficiency of rootstock and scion breeding and to develop improved rootstock and scion breeding lines to meet modern marketing and growing criteria. The scientific objectives of this contract were as follows: 1. Initiation of rootstock mapping and development of new techniques for rootstock selection To identify molecular markers linked to rootstock characters of interest, e.g. vigour, compatibility, drought tolerance, replicated segregating progenies need to be established and new screening techniques developed for generating phenotypic mapping data. 2. Identification and development of markers Markers that are currently available derived from scions, e.g. those linked to resistance to scab, mildew and aphids; need to be tested in rootstock and cider germplasm. Further markers, e.g. those linked to woolly apple aphid resistance, to the precursor gene for resistance to rosy leaf curling aphid, to columnar habit and to fruit quality, are necessary to increase the efficiency of the marker-assisted selection (MAS). 3. Transfer of marker technology The marker technology needs to be transferred to the Apple & Pear Breeding Club (APBC) for application within the industry-funded breeding programme. 4. Development and transfer of new breeding lines and novel material to the APBC New parental material will be developed exploiting East Malling Research genebank material to incorporate the ‘novel’ resistances that are becoming more important to ensure the sustainable production of top fruit. In addition, novel rootstock and scion breeding progenies, after initial screening, will be transferred to the APBC for final selection and trialling in order to fully exploit the material and technology developed within this Defra project. 5. Project review Progress and interaction with the APBC and the National Association of Cider Makers will be discussed annually with the Defra project officer. A rootstock mapping progeny was raised from the cross of M.27 (dwarfing) x M.116 (woolly aphid resistant) and seedlings were lined out and hard pruned. This will provide cuttings for the future replicated testing which is necessary for identifying molecular markers linked to quantitative rootstock traits. The seedlings were scored not only for woolly aphid resistance and virescence (a deleterious yellow leaf characteristic) but also for a set of 147 microsatellite markers. A framework genetic map was prepared on which virescence and woolly aphid resistance were located and which will be invaluable for the follow-on project WU0115 ‘Genomics tools for pre-selection of WUE in top fruit rootstocks’. A wide range of new rootstock progenies were raised using traditional Malling rootstocks and selections from other programmes and wild species. These are being evaluated with a new fast-track system and from the oldest progeny, AR86-1-20 x M.20, six selections have been identified for replication and trial. New markers have been mapped in apple that are linked to virescence, woolly apple aphid resistance, self-compatibility, columnar habit, weeping habit and susceptibility to the strobilurin fungicide Amistar which will be useful for the development of MAS in breeding programmes. We have successfully mapped the first marker for aphid resistance in pear. A series of new QTLs have also been mapped that are linked to fruit quality traits within the EU HiDRAS project. Industry-funded staff have been trained in marker technology and have used a number of markers to confirm the presence or absence of key traits, e.g. scab, mildew and aphid resistances, in advanced selection and parental material from the apple breeding programme. This background information is particularly useful to the breeder when devising new crossing programmes. New progenies and breeding lines developed with sustainability as a target have been passed to the industry-funded project for evaluation (11 apple rootstock progenies, eight pear rootstock progenies and over 1100 individuals pre-screened from 58 scion progenies). The additional focus on rootstocks has lead to a renewed interest from the UK fruit and cider industries and consequently improved links with the Horticulture Development Council (HDC). Our broadening of the genetic base of the rootstock collection will be useful in responding to climate change. Four papers have been published in peer-reviewed journals on apple mapping and marker development, three are submitted pending review and another 12 papers have been published or submitted to non peer-reviewed journals. Opportunities for technology transfer have been have been fully exploited with over 40 presentations and meetings attended with groups representing the scientific community, the fruit industry and the general public. Thus the project has made excellent progress in developing genetic resources – progenies, maps, markers and selection techniques – to underpin rootstock improvement, which will provide the foundation for the new project on ‘Genomics tools for pre-selection of WUE in top fruit rootstocks’ and has publicised these results at every opportunity. In addition it has developed valuable markers and breeding lines for exploitation via the APBC and the new East Malling Rootstock Club (EMRC). Both routes should benefit the UK grower by providing rootstocks and scions more suited to sustainable production with e.g. a range of disease and pest resistances and better drought tolerance.

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 scientific objectives of the contract

The acreage of dessert and culinary apples and pears in Britain is approximately 15,000 ha and produces a crop worth about £80 million wholesale. Imports are worth about £300 million. There is an additional 5,000 ha of cider apples supporting a cider industry valued at around £4.5 billion per year. Sustainable production of top-fruit in the UK is greatly dependent on any advances that can be made in the development of new improved scions and rootstocks to face the increasing competition from overseas. The majority of important characters for scion varieties are obvious, for example, resistance to pests and diseases, compact habit for ease of harvesting, and appearance, texture and flavour. It should be pointed out that pest and disease resistance are desirable for cider production; moreover, cider growers have expressed interest in columnar habit to aid mechanical harvesting. The effects of the rootstock on production are less widely appreciated. Currently one of the most important factors for a grower is the cost of labour; using a dwarfing rootstock can reduce the cost of pruning and also simplify picking. Apples for cider production are harvested by mechanically shaking the tree, so the anchorage properties of the rootstock and the strength of the scion/rootstock union both have to be considered. Other important rootstock characters are the induction of precocious and reliable cropping, ease of propagation, freedom from suckers and, of course, resistance to pests and diseases. Climate change and the weather over recent years has increased the demand for both drought tolerance and flood tolerance.

Breeding improved varieties of top-fruit (except cider apples) for UK growers is conducted at East Malling Research (EMR). As detailed later, top fruit breeding is a long and labour intensive process, with scions taking at minimum around 12-15 years before release and rootstocks up to 30 years. Breeding for rootstocks traditionally involves making controlled crosses, germinating seeds, raising a small number of seedlings (<1000 per year) under glass, and planting these in the field for selection. There are very few practical selection techniques for rootstocks; for example, assessment of percentage root-bark that was routinely used in the past as an indication of dwarfing can no longer be undertaken routinely as it is labour intensive. Following minimal early selection, two stages of long term trials (each taking around 10 years) are necessary in order to obtain orchard performance data. Breeding scions is rather more efficient as there are more selection techniques available for rejecting inferior seedlings at an early stage. However, these are time-consuming, and selecting for fruit quality cannot be undertaken until the seedlings have flowered about six years after germination. There has been a little work on breeding cider varieties at IACR Long Ashton, which has now closed.

Marker-assisted selection (MAS) promises to streamline and increase the efficiency of plant breeding programmes, potentially halving the time taken to develop a new rootstock. It involves identifying genetic variation, typically in protein or DNA sequences that can be detected readily in the laboratory that is linked to a specific phenotype. This is done by correlating the presence of the genetic variant with the phenotype in a segregating progeny. Subsequently, seedlings can be screened soon after germination for the presence of the markers indicating desirable characters. Considerable advances have been made in the development of MAS in apple at EMR (initially with EU EAGMAP and BBSRC 204K and, more recently, with Defra HH1029STF and EU DARE). However, that work concentrated on mapping and detecting markers for phenotypes of importance in dessert scions. It is now timely to implement MAS in scion breeding and initiate work that will allow MAS in rootstock and cider apple breeding.

In association with the development of MAS there is an opportunity to introduce certain novel useful characteristics into breeding lines, for example drought tolerance, which may become increasingly important with our changing climate.
This project capitalises on our current genetic expertise (including the new Defra/EU HiDRAS project) and breeding lines to increase the efficiency of the rootstock breeding and to develop improved rootstock and scion breeding lines to meet modern marketing and growing criteria. Breeding lines from this project feed directly into the industry-funded top fruit improvement programme, the APBC and its successor, the EMRC. Recent variety releases from the APBC include ‘Meridian’ (an apple scion in 2000), ‘Park Farm Pippin’ (an apple scion in 2002), ‘M.116’ (an apple rootstock in 2001) and ‘EMH’ (a quince rootstock for pear in 2001).
The scientific objectives of this contract were as follows:
1. Initiation of rootstock mapping and development of new techniques for rootstock selection

To identify molecular markers linked to rootstock characters of interest, e.g. vigour, compatibility, drought tolerance, replicated segregating progenies need to be established and new screening techniques developed for generating phenotypic mapping data.

2. Identification and development of markers

Markers that are currently available derived from scions, e.g. those linked to resistance to scab, mildew and aphids; need to be tested in rootstock and cider germplasm. Further markers, e.g. those linked to woolly apple aphid resistance, to the precursor gene for resistance to rosy leaf curling aphid, to columnar habit and to fruit quality, are necessary to increase the efficiency of the MAS.

3. Transfer of marker technology

The marker technology needs to be transferred to the APBC for application within the industry-funded breeding programme.

4. Development and transfer of new breeding lines and novel material to the APBC

New parental material will be developed exploiting EMR genebank material to incorporate the ‘novel’ resistances that are becoming more important to ensure the sustainable production of top fruit. In addition, novel rootstock and scion breeding progenies, after initial screening, will be transferred to the APBC for final selection and trialling in order to fully exploit the material and technology developed within this Defra project.

5. Project review

Progress and interaction with the APBC and the National Association of Cider Makers will be discussed annually with the Defra project officer.

Extent to which the objectives set out in the contract were met

All objectives were met in full and on time. Several milestones were reviewed with the project officer during the course of the project and updated where necessary.

Methods and results
Objective 1 – rootstock progenies

Establishment of segregating progenies of rootstocks

Crosses were made by hand pollination under glass of M.27 (dwarfing) x M.116 (woolly aphid resistant, moderate vigour) and of Mildew Immune Seedling (resistant to mildew and woolly aphid) x M.27 to raise progenies for mapping key traits; 308 and 1,637 seeds, respectively, were obtained and 300 of each sown and stratified.

Some 120 and 180 seedlings respectively of each of the mapping progenies M432 (M27 x M116) and M430 (Mildew Immune Seedling x M27) were planted in the nursery to establish trees for hedging prior to multiplication for replicated tests. The plants were pruned hard each winter to develop as hedge trees for provision of hardwood cuttings for replicated evaluation. The first batch of cuttings was successfully collected in winter 07/08 for rooting.
Progeny M432 was scored for the deleterious recessive character virescent (leaves yellow in spring, turning green in warmer weather), which is common in rootstock progenies and for which a marker would be useful to detect heterozygotes. The segregation was approximately 3:1. A replicate set of M432 was produced by grafting on to potted rootstocks in spring 2006. This was evaluated in the glasshouse for resistance to woolly apple aphid (Eriosoma lanigerum), a pest of rootstocks likely to become more of a problem as temperatures increase, by controlled inoculation. Approximately five aphids were placed on each plant up to three times in early summer and subsequent colonisation was monitored; individuals on which colonies developed were classed as susceptible. The segregation was approximately 1:1.
Potted seedlings of the rootstock progeny M430 were likewise scored in the glasshouse for resistance to woolly apple aphid and also for powdery mildew (Podosphaera leucotricha). The segregation in both cases was approximately 1:1.
Of the two mapping progenies, M432, a backcross, was selected as being the priority progeny for mapping due to the wide range of phenotypes expected. Leaf tissue was collected and frozen with liquid nitrogen. DNA was extracted from 188 individuals using the standard CTAB method. A set of 187 microsatellite (SSR) markers were chosen that were well-distributed across the ‘Fiesta’ x ‘Totem’ map. These were scored initially in M.27 and M.116 and a small set of seedlings. Then the 147 that segregated were scored in the full M432 progeny using fluorescently labelled SSR primers and multiplexed reactions whenever possible. The data were collected using an ABI 3100 Genetic Analyzer with an internal size standard (ROX™ or 500 LIZ™) using GENESCAN^â 3.7 and GENOTYPER^â 3.7 software (Applied Biosystems).
A framework genetic linkage map has been generated using the software JOINMAP® 4.0 (Van Ooijen 2006 and includes 141 SSRs (see Appendix 1). The virescent locus v mapped to linkage group 12 and the woolly apple aphid locus Er mapped to linkage group 8. As the initial backcross to produce the progeny was semi-compatible due to a common S (incompatibility) allele, the segregation of loci around the S locus was highly distorted. This was expected and would only be problematic if a trait of interest is closely linked to the S locus.

Development of new rootstock progenies

Throughout the five years of the project, a series of new rootstock progenies has been generated by controlled crosses to introduce novel resistances and other important characters into EMR’s breeding lines. Parental material includes Malus floribunda which is not only resistant to apple scab but is very early leafing and flowering and needs a lower winter chilling period than the Malling rootstocks, Geneva 202 (known to have multiple resistances), the ‘exotic’ Morioka rootstock JM7 (Malus sieboldii x M9), which has a novel source of aphid resistance and is reportedly very easy to propagate (Yoshida et al., 1998), P. amygaliformis (reputed to be drought tolerant) and P. betulaefolia which is thought to be tolerant of flooding (Lombard and Westwood, 1987).

The progenies include apple rootstocks M306 (AR86-1-20 x M.20), M345 (M.M.106 x ‘Totem’), M360 (AR86-1-20 x M.9), M480 (M.9 x M.116), M481 (M.9 x G202 (Geneva)), M508 (M.13 x JM7 (Morioka)), M545 (M.9 x Geneva 202), M546 (M.9 x JM7), M547 (M.9 x M. floribunda 821), M548 (M.13 x Geneva 202), M549 (M.13 x JM7) and pear rootstocks PQ37 (B617 x B627), PQ38 (QR708-36 o.p.), PQ39 (QR517-9 o.p.), PQ40 (B615 x ‘Kumloi’), PQ41 (B614 x Kumloi), PQ42 (B615 x P. amygaliformis), PQ43 (B616 x P. amygaliformis) and PQ44 (B622 x P. betulaefolia). Seedlings were raised, lined out in the nursery and then worked in situ to the columnar scion SA544-28 (or ‘Concorde’ for pear rootstocks) following the new rootstock assessment system. The progenies have been scored for crop, vigour and suckering over several seasons depending on their age.
Of the most advanced rootstock progeny, M306 (AR86-1-20 x M.20), six individuals were selected as having consistently good yield and few suckers and making smaller than average trees; these were cut back below the graft union so that shoots will grow out during 2008 that can be passed to the new EMRC as hardwood cuttings for establishing a trial.

Development of new screening techniques

Following discussions with EMR physiologists about methods for determining differences in water use of rootstocks, it was obvious that the level of replication required to map these traits, which were most likely to be quantitative traits, was not available with the material available in this project. However screening techniques for water use efficiency (WUE) which have been further developed as part of WU0107 (‘Determination of the response of strawberry to water-limited conditions, identification of QTLs associated with water use efficiency (WUE) and development of molecular markers for use in a genetic improvement programme’) will be applied to replicated plants of the mapping progeny M432 as part of the new project WU115 ‘Genomics tools for pre-selection of WUE in top fruit rootstocks’.

One technique, to measure stomatal conductance, was considered worth an initial test within this project. Here the relative rates of transpiration between the individuals of progeny M345 (M.M.106 x ‘Totem’) and of progeny M360 (AR86-1-20 x M.9) were measured using a porometer in mid-summer (see Fig. 1). These initial results indicate that, as expected, this trait has a continuous distribution.

M360

Number of individuals

stomatal conductance mmol²/s

Figure 1. Distribution of stomatal conductance in progeny M360.