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 GENESCANâ
3.7 and GENOTYPERâ
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
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.
Number of individuals
stomatal conductance mmol2/s
Figure 1. Distribution of stomatal conductance in progeny M360.