- Constitution of the core-collection of Venturia inaequalis. The collection comprises 319 strains from the 8 European countries involved in the DARE project, isolated on a range of 48 different Malus species and Malus x domestica cultivars. This range comprises the most cultivated clones in Europe, but also polygenic resistant cultivars, Vf resistant cultivars, and several Malus species considered as sources for scab and mildew resistance. This collection represents a great diversity of geographic and cultivars origins. The collection is kept by INRA-Angers and is open to the partners of the DARE project. - Test of the pathogenicity of a set of strains. A set of 39 strains from 7 countries and 28 cultivars was tested, comprising 18 strains isolated from susceptible cultivars, 13 from polygenic resistant cultivars and 8 from Vf resistant cultivars, in the countries where this resistance is broken. This test gave interesting information on the presence of strains virulent to the Vf gene in Europe. Additional information on the susceptibility of the host range was obtained, and insight into the virulence and aggressiveness of the European strains of V.inaequalis.
Evaluation of resistance level of 37 apple cultivars as regards numerous Podosphaera leucotricha inocula
To identify apple cultivars that show broad resistance towards different local inocula of the apple powdery mildew fungus Podosphaera leucotricha, a large experiment was set up at four European locations: Ahrensburg (Germany), Dresden-Pillnitz (Germany), Wädenswil (Switzerland) and East Malling (England). A list of 37 cultivars including some Malus species and their derivatives has been defined considering the traditional knowledge about their low susceptibility for powdery mildew. The mildew scoring data collected over three years of assessment in the field as well as under glasshouse conditions gave further confirmation that there is a high potential for selecting tolerant and resistant apple genotypes even within cultivated apples. As expected, most of the Malus species and derivatives are mildew resistant. But also some of the cultivars as for example 'Dülmener Rosenapfel' and 'Rote Sternrenette' were classified as "resistant". They could be used as basic material in breeding new apple varieties combining good fruit quality with a high level of powdery mildew resistance. The usage of cultivated apples as crossing parents instead of species or species-like breeding material may reduce time and costs of resistance breeding projects since the number of backcrosses necessary to reach a good fruit quality level would be considerably lower.
Creation of numerous progenies by crossing cultivars carrying broad-spectrum resistance to scab and/or powdery-mildew
The main objective of this part was to prepare new apple breeding material combining different types of resistant genes (major genes, QTLs ) against scab and powdery mildew. 61 crosses have been performed by 7 institutes: INRA, PRI, ETHZ-FAW, BAZ, HRI, DCA-BO, CRA. They represent various combinations of scab resistance genetic mechanisms : Vf x polygene, polygene x polygene, susceptible x polygene, Vf x Vr. Some crosses have also been performed to combine scab (Vf) and mildew resistance (Pl1 and Pl2). The scab resistant parents have been chosen from the result number 1143 of this project that screened 36 cultivars with a large range of scab strains. Some scab tests have just started at the end of the project. They confirm the polygenic nature of the resistance of some parents (Alkmene, Rote Sternrenette), which transmit very low amount of resistance in their progenies. This work is the first step toward new breeding strategies for durable resistance in apple. As shown in result number 1143, selection for partial resistance needs to develop new screening methods. Molecular markers are certainly useful tools for the breeders to get new cultivars, which combine durable resistance and high fruit quality standard.
The Fiesta x Discovery (FxD) progeny was tested at Plant RI and ETHZ/FAW. At Plant RI, 235 genotypes of FxD were grafted on MM106 by a commercial nursery in 1997; then two copies per genotype were transferred to the experimental station at Elst, the Netherlands. The presence of mildew was recorded on a 6-point scale (0=no symptoms to 5=severely diseased) at 8 different dates over 2 years (1999 till 2001). At ETHZ/FWA the FxD progeny was tested at Wädenswill (FAW). Grafts (MM106) were obtained from Plant RI. One copy per genotype was planted in summer 1999. Disease assessments were performed three times in 2000 and once in 2001 on a scale of 0 to 4. QTL-analysis: interval mapping QTL-analysis was performed using MapQTL (Van Ooijen et al., 2002); not any significant QTL could be identified (Lod>3.0) for any date of observation. QTL analysis: single marker analysis. Markers were entered at one at time to investigate whether an association existed between the marker and the disease score. Only markers with large F-values and consistent results over years were considered: significant F-values were obtained for markers belonging to 6 genomic regions. For those markers, QTL effects are all close to 0.5, i.e. half a unit on scale of the disease score. QTL analysis: inter QTL interactions. Four QTLs were identified that probably are involved in inter-QTL interactions. Linear regression analysis confirmed this. An interaction pattern among 3 QTLs indicates that in September high disease scores occur only when all three QTLs are simultaneously absent. The presence of just one favourable QTL-allele increased the level of resistance considerably. Addition of a second or even third QTL did not result in a statistically significant increase in resistance. 3 to 4 QTLs explained roughly around 90% of the estimated genetic variation of the May data (average of 2 years) and 50% of the September data. These are rough estimates since they depend severely on the accuracy on the estimations of the experimental variation, which is based on the variation of the reference cultivars, and on the validity of the identified interactions. They nevertheless indicate that we probably identified most of the QTLs involved in winter survival (scores in May), and some QTLs related to epidemic build up (scores in September).
This part of the project was devoted to the evaluation of consumer's preferences and market study for new resistant apple varieties. Consumer's preferences regarding sensory qualities of apples in new scab-resistant varieties have been evaluated in Switzerland and in France. In France, classification tests of new resistant varieties with regard to control varieties were also developed, as well as sensory profiling of these new varieties performed by professional panellists. According to the questioning, Swiss people mostly eat apple because they like them and because they are said to be healthy. Moreover, apples are an appreciated snack between meals, especially as they are very refreshing. Several tests allowed to classify Otava and Goldrush (two Vf-resistant cultivars) with regard to Golden Delicious, and Ariwa and Topaz (two other Vf-resistant cvrs) with regard to Royal Gala. The resistant varieties were generally well classified in comparison with the control varieties. The two new hybrids evaluated in France were well appreciated for both visual aspects and taste. Intentions of purchase for both hybrids were very high. Nevertheless, it appeared that sensory tests are very important before launching new apple varieties. Indeed, tasters are often polite and care must be taken to ask the right questions to obtain consumer's response without prompting their reply in any particular direction. The analytical parameters (such as firmness, total soluble solids, titrable acidity) are not very valuable for predicting the overall acceptance of apple varieties, and finally it is difficult to communicate about 'diseases resistance' to consumers.
A candidate gene analysis has been performed to identify and map NBS (nucleotide-binding site) or HcrVf resistance gene analogues (RGAs) in the apple genome. The aim is to try to characterise the biological function of major genes or QTLs of resistance through co-localisation of the detected RGAs and the previous resistance loci. Numerous NBS-RGAs have been cloned with degenerate primers corresponding to the conserved regions of already-cloned major resistance genes (N, L6, RPS2, RPP5) and mapped on several apple genetic maps as RFLP, CAPS or SSCP markers. Other RGAs have been mapped with the modified-AFLP technique, using primers corresponding to sequences of HcrVf genes (genes putatively corresponding to the major resistance gene Vf, whose cloning is under way). Several co-localisations between these RGAs and major resistance genes or resistance QTLs have been observed. It is especially the case for the two major genes Vd (on linkage group LG10) and Vg (LG12), for which two NBS-RGAs have been identified in their vicinity. These RGAs can be used as specific markers to follow these two genes in breeding generations.
Construction of a European saturated reference genetic map of the apple genome based on the 'Prima x Fiesta' progeny
Prima x Fiesta - Extension and saturation of the genetic map: For this progeny, a preliminar genetic map was already available at the beginning of the project (Maliepaard et al., 1998). This map was enriched with numerous new RFLP and AFLP markers, using the software JoinMap (Stam and van Ooijen, 1995). The map of the cross Prima x Fiesta consists of 17 linkage groups for each parental genome which is equal to the basic genome number. The map of Maliepaard et al. (1998) included a total of 294 markers (197 in Prima and 168 in Fiesta of which 70 appear on both maps) and covered 843.0 cM and 983.0 cM in Prima and Fiesta, respectively. To these parental maps 178 and 144 new markers were added respectively, of which 57 appear on both maps. The increased marker density revealed some previously undetected problematic regions of the map that could now be eliminated by re-scoring and re-mapping of the involved markers. Such regions were slightly reduced in length, indicating better fitting markers and less tension. Eight previous markers (5 RAPDs, 2 RFPL, 1 isozyme) were removed since they caused problems which could not be resolved. The new map shows a net increase of 132.1 cM for Prima and 188.8 cM for Fiesta, now spanning 975.1 and 1171.8 cM, respectively. The length of the individual linkage groups ranged from 39 cM to 98 cM with an average of 62 cM. The number of markers ranged from 12 to 41 with an average of 20. Various linkage groups could be extended by more than 20 cM, and the number of gaps (distance between successive markers larger than 20 cM) reduced from 11 to 7. One major inversion in marker order has been obtained in the lower part of linkage group P16 and various small inversions appeared throughout the map. New in the visual presentation of the map is the inclusion of: - The linkage phase of markers. - The molecular weights of the SSR, RFLP, and AFLP markers. - The restriction enzymes used in the generation of RFLP markers. This information allows to deduce which markers are on the same chromosome as a gene or QTL of interest, and to deduce the segregation type of a marker (which determines the accuracy by which it can be mapped (Maliepaard et al. 1997). The inclusion of restriction enzymes improves conditions to explore RFLP data.
New genetic markers linked to the apple gene Pl1 conferring major resistance to Podosphaera leucotricha
The objectives of this task were to evaluate powdery mildew resistance/susceptibility of several mapping progenies generated from crosses between susceptible and resistant genotypes and to develop molecular markers that could enhance the efficiency of resistance breeding. By crossing the susceptible variety 'Idared' with a Pl1- mildew resistant genotype, a large segregating progeny of about 400 individuals was produced and analysed phenotypically and genotypically. For the latter approach a marker-enrichment strategy based on the AFLP marker technique has been used to identify additional molecular markers that are linked with the Pl1 locus. The linkages between the resistance trait and the new molecular markers are tightly enough to allow a marker assisted breeding. SCAR markers have been developed that are technically more easily to screen in large breeding populations. The clear segregation patterns for resistance/susceptibility and the good correspondence between phenotypical and marker data found at the location Ahrensburg (Germany) was in agreement with the a priori hypothesis that Pl1 is controlled by a single major gene. However, considering phenotypic observations and marker data from breeding experiments in other countries, it seems likely that at least a second important gene has to be considered in particular genetic situations. In conclusion, the results regarding Pl1 markers and -genetics are important for apple breeders who are planning to involve molecular tools within the breeding process.
Characterisation of new virulences in Podosphaera leucotricha strains, and evaluation of genetic diversity of European strains
To prove the putative presence of physiological races of the apple powdery mildew fungus Podosphaera leucotricha, 31 monoconidial isolates representing six different European origins have been established in vitro. All strains were analysed by using an AFLP-based DNA fingerprinting technique. The genetic diversity calculated from the AFLP data of the 31monoconidial isolates was generally low but increased at larger geographic distance from Ahrensburg (Germany). A semi-sterile "detached leaf assay" has been chosen for pathogenic tests of six monoconidial isolates that have been selected due to their genetic distances determined by the molecular marker analysis. As a result of the inoculation tests, a set of seven apple cultivars was defined that enables the differentiation of five of the six monoconidial isolates due to their mildew infection patterns. Considering the molecular results together with the observations from the pathogenic tests, there is a high probability that at least a few powdery mildew races may be present in Europe. This information is very important for further resistance breeding strategies. Powdery mildew resistance has been based in the past mainly on a few monogenic resistances from Malus wild species. With the expanding number of mildew resistant apple cultivars there might be an increased risk for the breakdown of resistance due to a rapid development of new virulent races. As there are only few epidemiological data concerning apple powdery mildew available so far, the results regarding the genetic structure of a set of European fungal isolates are a first step in developing risk assessment models for this apple pathogen worldwide.
Evaluation of resistance level of 36 apple cultivars as regards numerous Venturia inaequalis strains or inocula
The aim of this part is to assess for the resistance status of a large list of apple cultivars regarding a wide range of scab strains in order to test their level of durable resistance. This collaborative work involved 6 partners (DCA, BAZ, ETH, CRA, INRA, PRI) who tested in greenhouse 36 cultivars in front of 14 various scab inocula: 6 obtained from collecting scabbed leaves in the fields of each institute, 5 monoconidial strains isolated and characterised by INRA before the start of the project and 3 other monoconidial strains obtained and characterised in the frame of the result number 1874 of this current project. The results showed a high diversity between the different inocula both for aggressiveness and virulence. A high range of resistance has been found in the tested cultivars: 9 local cultivars exhibited a wide spectrum of resistance: they could be used as potential parents for further breeding works on durable scab resistance. Considerable methodological knowledge has been gained in the evaluation of partial scab resistance using macro and microscopic assessments in greenhouse. Nevertheless further studies are still requested to confirm in the field these results and test if the behaviour of these scab partial resistant cultivars is the same on young and adult trees and between leaves and fruits. The new European Hidras should answer a part of these questions.
Identification and location of major genes and QTLs (Quantitative trait Loci) for resistance to various strains/inocula of Venturia inaequalis in several apple progenies
In addition to the saturated reference genetic map of Prima x Fiesta, four detailed genetic maps have been constructed for 4 connected progenies (Discovery x TN10-8, Fiesta x Discovery, Discovery x Prima, Durello di Forli x Fiesta) mainly with isozymes, AFLP and microsatellite markers. Each progeny contained at least 150 individuals. Discovery, TN10-8 and Durello di Forli are considered as scab resistant cultivars (partial resistance); Prima is a Vf-resistant cultivar which also carries the Vg gene (resistance to race 7 of Venturia inaequalis); Fiesta was initially considered as a susceptible cultivar. Two additional progenies were also used to map the two major scab resistance genes Vr and Vbj. Numerous pathological tests have been performed on the four first progenies, allowing the localisation of both major resistance genes and resistance QTLs (Quantitative Trait Loci). Certain tests were performed with strains isolated in another part of the project. Several major genes, such as Vbj, Vr, Vg or Vd are now located on the reference linkage groups and closely linked molecular markers are under development. Several hot spots for resistance (QTLs) have been identified, such as on linkage groups 17, 11, 2, 1, 3, 13 and 9 of the reference map. They all are likely to have biological validity since they have been identified in at least two independent data sets for resistance. Interestingly, clustering of major genes and QTLs have been identified for linkage groups 1, 2, and 11. A significant level of QTL interaction has been shown for many QTLs, so that, in several cases, a significant QTL could be efficient only when the favourable allele of another QTL was present. The genetic background may also influence the expression level of a QTL. Moreover, significant QTLs appeared to be rather strain specific: a QTL could be detected with some strains and not with other strains, or with a much lower effect. This in-depth analysis of the genetic bases of scab resistance highlights the complexity of the apple x Venturia inaequalis interaction, and should help to draw lines for future apple breeding strategies involving marker-assisted-selection.
The presence of strains of V. inaequalis virulent to the Vf gene was detected by two different approaches: - The assessment of scab on a set of differential hosts in a network of core-orchards planted in 8 DARE European countries. - The test of the pathogenicity of 39 strains, originating from different orchards of 7 countries, on a set of 8 differential hosts, in controlled conditions (growth-chamber). The results obtained during the DARE project, added to the previous publications relating the presence of races 6 and 7 in some European countries, permitted to map the distribution of these strains in Europe. This work showed that these strains are mainly present in the Northern part of Europe: Denmark, Northern Germany, The Netherlands, Belgium, Northern France and England. However, the presence of race 7 in the eastern part of Germany and Switzerland could indicate a spread in the eastern and central parts of Europe. Strains virulent to the Vf gene were not detected in Italy and Greece. Race 6, firstly only present in Germany, is now present or suspected in 4 countries. Strains cumulating the virulence of races 6 and 7 were found in The Netherlands. The presence of strains virulent to the Vf gene in commercial orchards in France, The Netherlands and Denmark strongly point out the need of strategies for the management of the Vf resistant cultivars.
An important amount of work have been made in the frame of this part: more than 1500 samples have been collected and observed during 4 years. In a first screening, 36 apple cultivars (same as in result 1143) were tested with 4 inocula: local inoculum and 3 monoconidial strains of Venturia inaequalis (races 1, 6 and 7). The tests were realized in the same conditions as in result 1143. The conidia germination was studied on foliar disks, 72 hours after inoculation. In addition to macroscopic observations, numerous scanning electron microscopy and histological studies were undertaken. The microscopical data give accurate assessment of the amount and the aspect of symptoms: presence, absence, abundance and aspect of subcuticular stroma, intensity and abnormalities of conidiogenesis, extension and intensity of host reactions. From this previous studies, 16 cultivars which showed typical resistance levels, have been selected: 13 cultivars with a partial resistance, 2 cultivars with a Vf resistance and a susceptible control. These cultivars have been inoculated with new strains isolated during this project (result n° 1874) : EU-B04, EU-D42, EU-NL24. Macroscopical and microscopical studies exhibit new symptoms of resistance and various level of strain aggressiveness. These selected cultivars allow to characterize a wide range of strains and to better understand the breakdown mechanisms. This knowledge should be very useful for the breeders and for further genetic studies on durable resistance.
Progenies of various parents carrying a single major resistance gene against scab have been created. The individuals with the Vf can and are easily selected through the presence of a single marker closely linked (Al07). The second most used major resistance gene currently is Vbj. The markers in this case are microsatellites (SSR). A single SSR is used in the first step (CH05e03), the presence of the Vbj is associated with the allele 150 bp at a distance of ca 1 cM. Verification is done by a second SSR (CH03d01) allele 115 at ca 6 cM on the other side of the gene in relation to the first marker. Other crosses with again Vf and Vr are currently genotyped with the marker for Vf and a Vr-linked marker. However the locus of resistance Vr currently identified does not correspond to the Vr resistance locus described prior and may correspond to another gene (putatively called Vrx). The individuals selected (25% of the progeny) were all scab resistant in glasshouse tests. They are or will be planted in the field and further observed. As far as possible selected individuals will have to be tested with the specific race capable of overcoming the resistance Vf. Combining major resistance genes against mildew is not yet feasible. Currently the available markers for two target genes Pl1 and Pl2 were only linked to the resistance in the original progeny where they were developed and mere not linked in other progenies as cross-tests by other partners revealed. As soon as the genetic background is clarified available progenies combining the two resistance will be analysed. The results demonstrate that marker assisted selection for pyramiding major resistance genes against scab is feasible. Single selections carrying both scab resistances will be available as soon as the trees will be flowering for further crosses and the tools for selecting the individuals maintaining both genes are also available.
New genetic markers linked to the apple genes Plw and Pld conferring major resistance to Podosphaera leucotricha
New markers were produced for two major genes for resistance to apple powdery mildew, caused by Podosphaera leucotricha (Pl-w and Pl-d): two SSRs and two SCARs for Pl-w and two SSRs and three AFLPs for Pl-d. Segregating progenies were screened for resistance to the disease and then scored genotypically with a range of different types of markers. The map position of Pl-w was confirmed to be on linkage group 8 and Pl-d was mapped for the first time onto linkage group 12 of the Maliepaard Prima x Fiesta map. These new markers can be used in breeding programmes via MAS to select individuals with either or both of the resistance genes. By adding two new mildew resistance genes to the current repertoire of R-genes mapped, the potential for selecting individuals with durable resistance is greatly improved. Such material will be of great benefit to the fruit industry and ultimately to consumers as chemical spray applications can be reduced.