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Objective 2 – marker development Marker transferability Seventeen cider apple varieties and 54 items from the rootstock germplasm collection were screened with 40 SSR markers known to be spaced across the apple map, and with COL (for columnar habit) and SDSSR (a very tightly linked marker for resistance to rosy leaf curling aphid, Dysaphis devecta), to establish their usability. The resulting data indicated that several have the gene for resistance to D. devecta, (M.11, M.18, M.20, M.25, M.M.101, M.M.102, M.M.104, M.M.105, M.M.106, M.M.109, M.M.111, M.M.115, ‘Northen Spy’, ‘Crab C', ‘Ellis bitter’ and ‘Yarlington Mill’). This was subsequently tested on a subset of 30 apple rootstocks and cider cultivars by inoculating with aphids in the glasshouse and scoring for resistance; the absence of reddening and failure of colonisation correlated with presence of the marker, demonstrating its robustness and applicability to survey work. ‘Fiesta’ x ‘Totem’ map EMR’s framework molecular map of ‘Fiesta’ x ‘Totem’, from the forerunner project HH1029STF, was further saturated within this project and the full 17 linkage groups were resolved. Seventeen microsatellite markers, multiplexed into four groups, received from the European HiDRAS project were screened on the progeny. A paper presenting the enlarged map (comprising approximately 250 microsatellite markers and mapping the gene for red tissue (Rt) for the first time) has been accepted for publication (Fernández- Fernández et al., 2008). In addition, the development of microsatellites from pear, that have also been mapped in ‘Fiesta’ x ‘Totem’, was written up and has been published (Fernández- Fernández et al., 2006; Inoue et al., 2007). Plant material and segregation data from the ‘Fiesta’ x ‘Totem’ progeny were supplied to the Defra-funded project on rosaceous genomics HH3724SSF in which isoenzyme and known function genes have been mapped in apple, cherry and strawberry so that the layout of the genomes of these three important crops can be compared using these anchor points. Such synteny between the genomes is very useful as data on the location of markers and candidate genes from each crop may be transferable and therefore exploitable in the others. A publication on the synteny of the three genera has been submitted (Sargent et al.). Markers for aphid resistances From the cross of ‘Cox’ (heterozygous for resistance to D. devecta) × ‘McIntosh’, 120 seedlings were inoculated with rosy leaf curling aphids in a gauze-house and scored for susceptibility, using the grading system of Alston & Briggs, 1968; unexpectedly, only 30 proved clearly resistant (and 5 were ‘intermediate’). Seedling DNA was amplified with primers for two markers tightly flanking the resistance gene Sd-1, SDSSR and D-DARM; 57 seedlings had the resistance markers. The occurrence of 22 susceptible seedlings having these markers was at first taken to support Alston’s hypothesis of a precursor gene, heterozygous in ‘Cox’ and ‘McIntosh’. Bulked DNA of susceptible seedlings with or without the markers was tested with a set of well spaced SSRs. However it was not possible to locate the ‘precursor’ gene and it may be that the behaviour of the aphids in colonising seedlings with the marker late in the season was anomalous. Apple crosses made to develop more markers included ‘Telamon’ × 3760, × ‘Florina’ and × ‘Ontario’, three different sources of resistance to rosy apple aphid (Dysaphis plantaginea) which gave 265, 275 and 405 seedlings respectively. These progenies were raised; 120 seedlings of each were inoculated with aphids and scored for colonisation and symptoms. The scoring in 2005 was hampered by contamination of the inoculum with another species. Fortunately, entomological colleagues on project HH3125STF agreed to undertake subsequent inoculations. The crosses were inoculated with four adult D. plantaginea in June 2006 and 2007. Three days after inoculation the plants were scored for aphid numbers and damage (leaf curl). Those plants that had lost aphids were reinoculated back up to four. A week later all the plants were scored again and reinoculated if necessary as before. The plants were then scored for the third and final time a week later. Mildew was a constant problem throughout the tests. Plants with mildew do not support aphid growth, so scoring plants for genuine resistance often becomes difficult. The seedlings were scored as susceptible (many aphids and leaf curl), intermediate (few aphids and no leaf curl) and resistant (no aphids and no leaf curl). Only plants that had a consistent score over the two years are reported. The ‘Florina’ cross showed no indication of resistance, with all the seedlings having large aphid colonies and leaf curl. This result concurs with a current Defra entomology project at EMR (HH3125STF) that shows ‘Florina’ to show no signs of resistance to the English D. plantaginea, although being sold in France as being resistant to French D. plantaginea. The Ontario cross was scored as 32 susceptible, 15 intermediate and 7 resistant; a 4:2:1 ratio. The 3760 cross proved to be more complicated. Resistance in 3760 is traditionally scored as a hypersensitive shoot tip death resulting in no aphid growth, hence this resistance has not been exploited in apple breeding. However, these seedlings were scored as resistant (tip dead, no aphids) 13, benign resistant (tip alive, no aphids) 17 and susceptible (many aphids, tip alive, and usually leaf curl) 32. This might indicate that a resistance mechanism may be usable without confounding tip death. This data will be useful for QTL mapping. In pear, 117 seeds were produced from a controlled cross between ‘Comice’ and Pyrus nivalis for mapping the resistance to pear-bedstraw aphid (Dysaphis pyri). The resulting progeny (P603) was screened for resistance in the glasshouse by controlled inoculation. A bulk segregant approach using a panel of well-spaced SSRs indicated that the gene for resistance was located at the bottom of linkage group 9 of the pear map Yamamoto et al., 2002, 2.3cM from the microsatellite NH006b. However, further analysis indicated that the gene for resistance was actually located on linkage group 17, not 9 as previously thought. These two linkage groups are very similar as a result of duplication of a section of chromosome when the Pomoideae (including both Malus and Pyrus) arose via a polyploidy event. The final position was resolved using different alleles of the same microsatellites which map to the different linkage groups. A paper is in preparation describing this work. Other characters Two seedling progenies, M377 (‘McIntosh’ (very susceptible) x ‘Golden Delicious’) and M403 (‘Golden’ x ‘Cox’ (moderately susceptible)), each of approximately 90 seedlings, were scored for susceptibility to application of the strobilurin fungicide azoxystrobin (tradename ‘Amistar’). Each segregated ca. 1:1 indicating a dominant gene for susceptibility in each case. Following a preliminary study, both were scored for six microsatellite loci from Linkage Group 12 of EMR’s ‘Fiesta’ x ‘Totem’ map: the two different resistances appeared to map to the same locus co-segregating with microsatellite GD127. Interestingly, analysis of a progeny from the cross ‘McIntosh’ x ‘Cox’ showed that the moderate susceptibility of ‘Cox’ is dominant to the strong susceptibility of ‘McIntosh’. A paper has been submitted reporting the mapping of the dominant gene conferring susceptibility in ‘Cox’ and ‘McIntosh’ (Tobutt et al.). A bulk segregant approach was initiated for investigating other traits of agronomic interest, including weeping habit, in two other progenies each of approximately 70 seedlings, M172 (‘Wijcik’ x ‘Red Jade’) and M140 (‘Elise Rathke’ x ‘Pink Pearl’). Using this technique, weeping habit from both sources has been mapped to linkage group 13, with the microsatellites GD147/161 and NH009b as flanking markers. The gene(s) for stripey red fruit versus green in the former progeny and for solid red colour versus blushed in the latter has also been mapped with a similar approach. It maps very close to microsatellites CH01h02z and EMPc115 on linkage group 9. Discussions were initiated with the cider industry to develop the necessary techniques for the assessment of phenolics. A preliminary screen was made on fruit from ‘Fiesta’, ‘Totem’ and eight seedlings by Reading Scientific Services Limited using HPLC; only minor qualitative differences were seen. Due to the lack of variation in this trait, it was agreed with the Defra Project Officer that this milestone should be replaced by one on columnar habit. Columnar habit Preliminary work showed that the SCAR marker SCB82670 reported by a Korean group to be linked to the gene for columnar habit (Co) – of interest to cider and juice growers as it offers scope for mechanical harvesting – is unreliable. Thus, though present in ‘Tuscan’, the columnar parent of the progeny in which it was developed, the SCAR was absent from ‘McIntosh Wijcik’, the original donor of the columnar trait, and from ‘Totem’, the columnar parent of our scion mapping progeny. The microsatellite markers CH03d11 and EMPc105, found to be tightly linked to Co on linkage group 10 of the ‘Fiesta’ x ‘Totem’ map, were scored in another progeny segregating for columnar habit, ‘McIntosh Wijcik’ x ‘Red Jade’, and the genetic distances of approximately 3cM confirmed. The 122bp allele from CH03d11 amplified in 10 out of 11 columnar cultivars or selections screened, the exception being ‘Charlotte’, and the 160bp allele from EMPc105 was found in all. Another SCAR marker, SCAR682, reported by a Chinese group, amplified the fragment linked to Co in the same 10 out of 11 samples. The inheritance by ‘Charlotte’ of the other CH03d11 allele from ‘Wijcik’, 124bp, and the absence of SCAR682 marker in this cultivar, indicates a recombination event. In breeding lines derived from ‘Charlotte’, the SCAR will be useless but the SSR can still be useful. A columnar cultivar of unknown origin, ‘Elegance’, had the same fingerprint for 10 SSRs from linkage group 10 as ‘Wijcik’ and is presumably a synonym. Self-compatibility Analysis of the self-compatible ‘Cox’ mutation ‘Queen Cox SF18’ and the progeny ‘Wijcik’ x ‘Queen Cox SF18’ showed that the self-compatibility is due to a duplication and translocation of the incompatibility allele S5 so that half of the pollen is heteroallelic (S5S9) and thus self-compatible because of competitive interaction; the other half of the pollen just has the allele S5 and is self-incompatible. So, self-compatible seedlings that do not need a pollinator can be selected in this breeding line using primers for the S9 allele. Additional segregating progenies produced Crosses made to segregate for key characters for future marker development included M166-11 x ‘No Pip’ (seedlessness), ‘Telamon’ x ‘Self-fertile Cox’ Clone 7 (self-fertility), M244-17 x ‘Reinette d’Espagne (false)’ (total lack of anthocyanin), ‘Rev. Wilks’ selfed (albino) and ‘Rev. Wilks’ x ‘Early Victoria’ (pale green lethal seedlings). Microsatellites for these last two deleterious recessive traits are desirable so that they can be eliminated from breeding lines. Other crosses made to enable the mapping of key traits included: Malus floribunda 821, the source of Vf scab (Venturia inaequalis) resistance, x ‘Fiesta’ to produce a progeny segregating for the polyphenolic compound sieboldin, which has been proposed as a component of resistance; ‘Anna’ (very low winter-chilling requirement) x ‘Feuillemort’ (very high winter-chilling requirement); and ‘McIntosh’ x ‘Adams Pearmain’, which is expected to segregate for dwarf seedlings. HiDRAS The EU Framework V project HiDRAS ran from 1.01.2003 to 30.09.07 and was a partnership of 11 research institutes representing eight countries (www.hidras.unimi.it/index.html). The EMR component was funded by the EU with matching funding from this Defra project. The principal aim of the HiDRAS project was to identify genetic factors controlling apple fruit quality with the objective of increasing the fruit quality of disease resistant apples and therefore their acceptability and diffusion, leading to a reduction in the use of fungicides. HiDRAS was based on an innovative approach to identify the genetic loci controlling the phenotypic variation of the most important components of fruit quality using pedigree-based analysis. This follows the segregation of specific chromosomal regions with highly polymorphic co-dominant microsatellite (SSR) markers in related cultivars, breeding selections and small progenies. This ‘identity by descent’ approach allows the detection of QTLs using the new software developed within HiDRAS. HiDRAS was an ambitious project in which over 1800 inter-related and progenitor individuals were genotyped with a genome-spanning set of 83 SSR markers and phenotyped for a range of fruit quality and disease resistance traits. EMR played a major part in this section of the project by genotyping all the individuals with 17 of the SSRs (18 loci), the majority of which were developed into three multiplexes. Many of the SSRs were novel, having been identified as part of HiDRAS. Using software developed as part of the project, several QTLs for fruit quality traits have been mapped such as acidity, texture and sweetness. Preliminary tests were run on the QTLs in different genetic backgrounds and different environments. A fully genotyped set of approximately 70 common progenitor cultivars were identified that were true-to-type and consistent with their known pedigrees. Such a set would be of value to add to the UK National Fruit Collections. Other areas within the project included the identification of candidate genes for texture and ripening and a range of consumer and sensory tests focussing on disease resistant varieties. Results from HiDRAS occupied a session of the 2007 Eucarpia fruit section Symposium. Several publications have already been published or submitted to refereed journals (e.g. Patocchi et al., submitted) and a number of further publications are planned. Data from the project are held in a specially designed database which will be opened in stages to the public following the completion of data mining by the partners. The principal benefits of this work for HH3604STF were: i. development of novel SSRs which could be mapped on to the EMR reference apple maps for scions and rootstocks; ii. experience gained in the development of multiplexing SSRs; iii. experience of working with the new QTL mapping software, and iv. identification of a number of useful QTLs. Objective 3 – marker technology Following discussion with the Policy Group of the APBC regarding the implementation of marker-assisted selection, training in marker technology was initiated for members of staff funded by the APBC and subsequently the EMRC e.g. DNA extraction, PCR and analysis of SSRs. This has continued. Staff funded by the APBC have successfully characterised 22 important parents and 14 progenitor varieties for molecular markers linked with four key traits, resistance to scab (V. inaequalis) and rosy leaf-curling aphid (D. devecta), two different sources of resistance to mildew (P. leucotricha) and the low ethylene trait associated with long storability (ACC synthase). A summary of these results from 12 important parents is shown in Table 1. Here, the selections are listed together with the proposed genes that were possible from their parentage. In each case, the positive allele or allele combination that is associated with the trait is highlighted with a shaded cell. In the majority of cases, where there had been selection for the trait by the breeder (primarily for scab and mildew resistance), the selection is shown to have the associated alleles. However, there are three examples (highlighted by underlining), where the markers do not confirm the presence of the gene. There are several possible explanations for this: a. the markers were not linked tightly enough to the trait; b. the trait could be determined by more than one gene; c. the breeders’ selection techniques were not infallible; d. the parentage of the selection is incorrect (not impossible when unbagged crosses are made). Table 1. Results of marker screening of 12 important breeding parents for four key traits with five molecular markers
Twenty of these parental selections have also been characterised for the presence of the first 23 QTLs linked to acidity and firmness that were identified in the final year of the EU-funded HiDRAS project. This information will be of considerable value when designing future crossing programmes. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||