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Climate change with warmer winter and spring temperatures poses major challenges to apple fruit production. Long-term observations confirm the trend toward earlier flowering, which leads to an increased risk of frost damage. New breeding strategies are needed to generate cultivars that are able to stay largely unaffected by warmer temperatures. Recently, epigenetic variation has been proposed as a new resource for breeding purposes and seems suitable in principle for apple breeding. However, to serve as a new resource for apple breeding, it is necessary to clarify whether epigenetic variation can be induced by the environment, whether it can create phenotypic variation, and whether this variation is stable across generations. In this brief review, we summarize the impact of climate change on the timing of apple phenology, highlight how epigenetic variation can potentially support novel breeding strategies, and point out important features of epigenetic variation that are required for its application in breeding programs.

Pyramiding different fire blight resistance genes and QTLs in future apple cultivars is the most eco-friendly way to combat this disease. Identification of strong fire blight resistance donors, and introgression of their resistance into apple breeding material are a continuing effort of breeding programs. Thus, enormous effort is been put into breeding research to understand host – pathogen interactions and mechanisms of resistance found in Malus. The crabapple Malus fusca (accession MAL0045) is highly resistant to fire blight, and although resistance is strain-dependent, resistance of MAL0045 is not overcome by any known strain of Erwinia amylovora to date. A strong fire blight resistance locus (FB_Mfu10) was fine mapped to an interval of 0.33 Centimorgan (cM) on linkage group (LG) 10 of MAL0045 using 1888 progenies. Subsequently, a single bacterial artificial chromosome (BAC) clone (46H22), which harbours FB_Mfu10-resistance alleles, was identified in a MAL0045 BAC library and sequenced using MiSeq illumina leading to the assembly of 45 contigs. Analyses of the sequence of 46H22 led to the identification of a receptor-like kinase candidate gene. Here, we report about resequencing 46H22 using MinION Oxford Nanopore and successfully assembled the sequences into a single contig, which allowed for identifying additional candidate genes.

Winter dormancy – a period of low metabolic activity and no visible growth – appears as an adaptation to harsh winter conditions and can be divided into different phases. It is tightly controlled by environmental cues, with ambient temperature playing a major role. During endodormancy, a cultivar-specific amount of cold needs to be perceived, and during ecodormancy, heat hours accumulate before bud burst and anthesis in spring. Expression analysis, performed in several key fruit tree species, proved to be very useful in elucidating the molecular control of onset and release of dormancy. However, the time resolution of these experiments has been limited. Therefore, in this study, dense time-series expression analysis was conducted for 40 candidate genes involved in dormancy control, under the cool-temperate climate conditions in Dresden. Samples were taken from the cultivars ‘Pinova’ and ‘Gala,’ which differ in flowering time. The set of candidate genes included well-established dormancy genes such as DAM genes, MdFLC-like, MdICE1, MdPRE 1, and MdPIF4. Furthermore, we tested genes from dormancy-associated pathways including the brassinosteroid, gibberellic acid, abscisic acid (ABA), cytokinin response, and respiratory stress pathways. The expression patterns of well-established dormancy genes were confirmed and could be associated with specific dormancy phases. In addition, less well-known transcription factors and genes of the ABA signaling pathway showed associations with dormancy progression. The three ABA signaling genes HAB1_chr15, HAI3, and ABF2 showed a local minimum of gene expression in proximity of the endodormancy to ecodormancy transition. The number of sampling points allowed us to correlate expression values with temperature data, which revealed significant correlations of ambient temperature with the expression of the Malus domestica genes MdICE1, MdPIF4, MdFLC-like, HAB1chr15, and the type-B cytokinin response regulator BRR9. Interestingly, the slope of the linear correlation of temperature with the expression of MdPIF4 differed between cultivars. Whether the strength of inducibility of MdPIF4 expression by low temperature differs between the ‘Pinova’ and ‘Gala’ alleles needs to be tested further.

A set of 680 apple cultivars from the Fruit Gene bank in Dresden Pillnitz was evaluated for the incidence of powdery mildew and scab in two consecutive years. The incidence of both scab and powdery mildew increased significantly in the second year. Sixty and 43 cultivars with very low incidence in both years of scab and powdery mildew, respectively, were analysed with molecular markers linked to known resistance genes. Thirty-five cultivars were identified to express alleles or combinations of alleles linked to Rvi2, Rvi4, Rvi6, Rvi13, Rvi14, or Rvi17. Twenty of them, modern as well as a few traditional cultivars known before the introduction or Rvi6 from Malus floribunda 821, amplified the 159 bp fragment of marker CH_Vf1 that is linked to Rvi6. Alleles linked to Pl1, Pld, or Plm were expressed from five cultivars resistant to powdery mildew. Eleven cultivars were identified to have very low susceptibility to both powdery mildew and scab. The information on resistance/susceptibility of fruit genetic resources towards economically important diseases is important for breeding and for replanting traditional cultivars. Furthermore, our work provides a well-defined basis for the discovery of undescribed, new scab, and powdery mildew resistance.

Malus ×arnoldiana accession MAL0004 has been found to be resistant to moderately and highly virulent strains of the fire blight causal pathogen – the Gram-negative bacterium, Erwinia amylovora. Genetic analyses with an F1 segregating population derived from crossing the highly susceptible apple cultivar ‘Idared’ and MAL0004 led to the detection and mapping of the fire blight resistance locus of M. ×arnoldiana to linkage group (LG)12 (FB_Mar12). FB_Mar12 mapped at the distal end of LG12 below the apple SSR Hi07f01 in an interval of approximately 6 cM (Centimorgan), where both the fire blight resistance loci of M. floribunda 821 and ‘Evereste’ were located. We fine mapped the region containing FB_Mar12 using 892 progenies. Mining of the region of interest (ROI) on the ‘Golden Delicious’ doubled haploid genome (GDDH13) identified the presence of 2.3 Mb (megabases) in the homologous region. Of 40 primer pairs designed within this region, 20 were polymorphic and nine were mapped, leading to the identification of 24 significant recombinant individuals whose phenotypes were informative in determining the precise position of the locus within a 0.57 cM interval. Analyses of tightly linked marker sequences on the M. baccata draft genome revealed scaffolds of interest putatively harboring the resistance loci of M. ×arnoldiana, a hybrid between M. baccata and M. floribunda. Open reading frame (ORF) analyses led to the prediction of first fire blight resistance candidate genes with serine/threonine kinase and leucine-rich repeat domains, including homologs of previously identified ‘Evereste’ candidate genes. We discuss the implications of these results on breeding for resistance to fire blight.

Malus domestica (apple) is one of the most important fruit crops worldwide. Fire blight, caused by Erwinia amylovora, is one of the most destructive bacterial diseases that impacts apple production systems worldwide. Although it is possible to manage fire blight using antibiotics such as streptomycin, kasugamycin or oxytetracycline, the quest for sustainable and eco-friendly production makes breeding for fire blight resistance the most promising and desirable approach. Breeding for resistance is a long, resource-intensive process due to the high susceptibility of most commercial apple cultivars, and the fact that most resistance sources being characterized are from wild genetic backgrounds with unpalatable fruits, and apple’s long generation times. Nevertheless, establishment of pre-breeding materials is crucial. This review highlights the status of breeding for fire blight resistance in Malus, taking into account, 1) major and minor resistance sources and their interaction with E. amylovora, 2) progress and challenges associated with using wild species as resistance sources, 3) progress and challenges associated with using elite cultivars as resistance sources, 4) advances in biotechnology for use in enhancing the production of durable fire blight resistant cultivars.

International audience; Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018 nearly 9000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14 and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, hence have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for the breeding, but their use is recommended only in extended pyramids of (≥3) resistance genes.

The Gram-negative bacterium Erwinia amylovora (Burrill) Winslow. et al., causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that functions to translocate effector proteins into plant cells that collectively function to suppress host defenses and enable pathogenesis. Until now, there is only a limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. In this study, we tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent Erwinia amylovora strains, which are assumed to influence the host resistance response of fire blight-resistant Malus species. Our findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene-for-gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple ‘Evereste’ and the wild species M. floribunda 821. Our results provide new insights into the host-pathogen-interactions between Malus sp. – Erwinia amylovora. This article is protected by copyright. All rights reserved.

The apple cultivar Regia, bred in Dresden-Pillnitz, Germany, is one of the few cultivars originating from a different scab (Venturia inaequalis) resistance background than Malus floribunda 821, the source of Rvi6. Cv. Regia is a descendant of Russian apple R12740-7A and has been proved to contain the two scab resistance genes Rvi2 and Rvi4. The cultivar itself has been grown in Dresden-Pillnitz since the early 1970s, while seedling populations derived from cv. Regia have been raised in fungicide-free plots since at least 1982. In 2011, small scab lesions were found for the first time on leaves of cv. Regia trees in an experimental unsprayed orchard in Dresden-Pillnitz. Single spore isolate Regia2 (R2) was cultured from these scab lesions and propagated, and then inoculated on scions of the VINQUEST scab differential host set comprising host (0) to host (15), host (17) and cv. Regia, grafted on rootstock M9, in a greenhouse. Strong sporulation was observed on hosts (0), (1), (2), (8), (9), (10) and cv. Regia, and weak sporulation on hosts (3), (4), (13) and (17), suggesting that the new isolate possesses the respective virulences. The implications of these results are discussed in relation to the natural infection of the differential hosts in the orchard and the artificial inoculation of the grafted differential hosts with inoculum gained from in vitro culture of R2.

Background Although the most common path of infection for fire blight, a severe bacterial disease on apple, is via host plant flowers, quantitative trait loci (QTLs) for fire blight resistance to date have exclusively been mapped following shoot inoculation. It is not known whether the same mechanism underlies flower and shoot resistance. Results We report the detection of a fire blight resistance QTL following independent artificial inoculation of flowers and shoots on two F1 segregating populations derived from crossing resistant Malus ×robusta 5 (Mr5) with susceptible ‘Idared’ and ‘Royal Gala’ in experimental orchards in Germany and New Zealand, respectively. QTL mapping of phenotypic datasets from artificial flower inoculation of the ‘Idared’ × Mr5 population with Erwinia amylovora over several years, and of the ‘Royal Gala’ × Mr5 population in a single year, revealed a single major QTL controlling floral fire blight resistance on linkage group 3 (LG3) of Mr5. This QTL corresponds to the QTL on LG3 reported previously for the ‘Idared’ × Mr5 and an ‘M9’ × Mr5 population following shoot inoculation in the glasshouse. Interval mapping of phenotypic data from shoot inoculations of subsets from both flower resistance populations re-confirmed that the resistance QTL is in the same position on LG3 of Mr5 as that for flower inoculation. These results provide strong evidence that fire blight resistance in Mr5 is controlled by a major QTL on LG3, independently of the mode of infection, rootstock and environment. Conclusions This study demonstrates for the first time that resistance to fire blight caused by Erwinia amylovora is independent of the mode of inoculation at least in Malus ×robusta 5.

The bacterium, Erwinia amylovora, deposits effector proteins such as AvrRpt2EA into hosts through the type III secretion pathogenicity island to cause fire blight in susceptible Malus genotypes. A single nucleotide polymorphism in the AvrRpt2EA effector plays a key role in pathogen virulence on Malus hosts by exchanging one cysteine to serine in the effector protein sequence. Fire blight resistance quantitative trait loci (QTLs) were detected in a few apple cultivars and wild Malus genotypes with the resistance of wild apples generally found to be stronger than their domestic relatives. The only candidate and functionally analyzed fire blight resistance genes proposed are from wild apple genotypes. Nevertheless, the aforementioned AvrRpt2EA SNP and a couple of effector mutants of E. amylovora are responsible for the breakdown of resistance from a few Malus donors including detected QTLs and underlying R-genes. This review summarizes a key finding related to the molecular basis underpinning an aspect of virulence of E. amylovora on Malus genotypes, as well as mechanisms of host recognition and specificity, and their implications on the results of genetic mapping and phenotypic studies within the last 5-6 years. Although the knowledge gained has improved our understanding of the Malus-E. amylovora system, more research is required to fully grasp the resistance mechanisms in this genus especially as they pertain to direct interactions with pathogen effectors.

Main conclusion Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents. Electronic supplementary material The online version of this article (doi:10.1007/s00425-016-2475-9) contains supplementary material, which is available to authorized users.

Eighty-two genotypes of Rubus available in germplasm collections, nurseries and home gardens were collected and evaluated using a set of 16 simple sequence repeat (SSR) markers to estimate the level of genetic diversity and relatedness of the germplasm and for testing them on trueness-to-type. Each of the 16 SSRs was successful in amplifying alleles from most genotypes. Fifteen of the markers produced polymorphic bands, whereas marker RhM023 was monomorphic. The polymorphic information content among genotypes varied from 0.056 to 0.83 with an average of 0.348. A neighbor-joining analysis allocated the genotypes to four major clusters containing 11, 24, 39 and eight genotypes, respectively. Cluster I consists of floricane-fruiting cultivars originating from the Scottish and/or British breeding programs or cultivars which have those cultivars in their pedigree. Cluster II included cultivars that have ‘Autumn Bliss’ or ‘Tulameen’ in their pedigree. Cluster III consists of summer-bearing raspberry cultivars, some primocane-fruiting cultivars, and a few intermediate summer-fall-bearing types. Cluster IV consists of the blackberry ‘Navaho’ (R. fruticosus L.), the interspecific hybrid ‘Dorman Red’ and a few other raspberry varieties. A number of yellow fruited varieties was dispersed on three different clusters suggesting a convergent evolution of this trait. The pedigree of several genotypes could be confirmed using a Pedimap based approach, whereas other cultivars were found to be genetically identical. The results disclose the alarming narrow genetic base of Rubus resources in Germany. Broadening of this base is urgently needed.

A total of 188 German and 28 Romanian pear accessions were evaluated using flow cytometry and molecular markers to detect homonymous and synonymous genotypes, triploids, which are not useful for breeding, and genotypes having favorable traits for breeding. Genotyping was done using a set of 11 simple sequence repeat markers proposed by the European Cooperative Program for Plant Genetic Resources, along with a standard set of six reference pear genotypes used in previous studies. The observed number of alleles ranged between 21 and 38 per locus. Twenty-three accessions could be confirmed as triploids. Because of the expected cross-incompatibility they are only of limited interest for breeding. The study confirmed that 180 of the 188 tested German accessions represent unique genotypes, while some accessions were found to be genetically identical. In parallel additional markers, which are known to be linked to resistance/susceptibility to major pathogens (e.g. black spot, aphid, fire blight, and apple scab), were applied to the German accessions. The comparative screening of the Romanian pear genetic resources demonstrated that only a few accessions with identical/very similar names to German accessions really have an identical fingerprint. Mislabeling and homonymous designation of different genotypes seems to occur frequently in different collections.

The development of high-quality cultivars, with durable disease resistance, is a major objective of apple breeding. The selection procedures of modern breeding programs no longer rely exclusively on phenotypic criteria but include marker-assisted breeding (MAB). Currently, molecular markers linked to several resistance genes and quantitative trait loci (QTLs) are available. In this study, we focused on markers available for resistance breeding against the major diseases scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha), and fire blight (Erwinia amylovora). One approach proposed to achieve durable resistance is the pyramiding of functionally different resistance genes against the same pathogen. This approach can be complemented with the incorporation of resistance genes against other pathogens. The resulting resistant apple cultivars would contribute considerably to low-input, sustainable, fruit production. Furthermore, apple cultivars can be developed carrying homozygous allele sets of specific resistance genes, and these genotypes can be used as parents for further crosses. Due to the ensured inheritance of the resistance genes to the progeny, MAB for these genes will become superfluous. In this study, we developed elite apple plants which are homozygous for three different scab resistance genes, Rvi6, Rvi2, and Rvi4. Furthermore, these apple selections tested positive for a resistance gene against powdery mildew (Pl1 or Pl2), and the FBF7 QTL from ‘Fiesta’ for enhanced fire blight resistance. Selected progeny plants were tested for their fire blight resistance after artificial shoot inoculation and evaluated for tree and fruit characteristics.

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain—Ea3049, and the avrRpt2EA mutant—ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains., Molecular Breeding, 38 (8), ISSN:1380-3743, ISSN:1572-9788

Phenotypic and genotypic characterization of existing sour and duke cherry cultivars provide information for use by breeders in developing cultivars with improved qualities and for germplasm management to optimise the conserved diversity. This study examined 79 sour and duke cherry cultivars of the Fruit Genebank of the Institute for Breeding Research on Fruit Crops, Dresden-Pillnitz. For morphological analysis, 23 descriptors of the tree, the flower or the fruit, and three phenological characters were recorded to detect similarities and differences among the cultivars. Most of the characteristics exhibited a high degree of variability. Principal component analysis revealed that the first three components were responsible for 48 % of the phenotypic variability. Color descriptors as well as the fruit size and weight, the organoleptic quality and the two descriptors related to the abscission between fruit and stalk were the most effective parameters to discriminate genotypes. The genetic analysis was performed using six microsatellite markers recommended by the European Cooperative Program for Plant Genetic Resources. The average number of the effective alleles was 7.11 and the expected heterozygosity ranged from 0.78 to 0.89. The highest expected heterozygosity was achieved with the primer pairs EMPaS10 and EMPaS12. The tree comparison (‘bipartition distance’) estimated by the two data sets, morphological and genetic, revealed a tree distance of 0.669. Apparently, more research is needed for combining and jointly interpreting phenotypic and genetic marker data in diversity analysis for plant breeding and germplasm management.

Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, is the most important bacterial disease affecting apple (Malus × domestica) and pear (Pyrus communis) production. The use of antibiotic treatment, though effective to some degree, is forbidden or strictly regulated in many European countries, and hence an alternative means of control is essential. The planting of fire blight-resistant cultivars seems to be a highly feasible strategy. In this study, we explored a segregating population derived from a cross between the wild apple species Malus fusca and the M. × domestica cultivar Idared. F1 progenies used for mapping were artificially inoculated with Erwinia amylovora strain Ea222_JKI at a concentration of 109 cfu/ml in three different years. The averages of percentage lesion length of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal–Wallis analysis was used to determine marker–phenotype association and revealed a linkage group with Diversity Arrays Technology (DArT) markers significantly linked with fire blight. After locating the positions of the DArT markers on the Golden Delicious genome, simple sequence repeat (SSR) markers were developed from chromosome 10 to replace the DArT markers and to determine the quantitative trait locus (QTL) region. Multiple QTL mapping (MQM) revealed a strong QTL (Mfu10) on linkage group 10 of M. fusca explaining about 65.6 % of the phenotypic variation. This is the first report on a fire blight resistance QTL of M. fusca.

A collection of 337 Malus accessions representing 46 species of the Fruit Genebank in Dresden-Pillnitz, Germany, were evaluated for 64 morphological and phenological traits to proof the classification of the accession and to analyze genetic relationships. Morphological characteristics were determined for the whole tree, shoots, leaves, flowers and fruits. Data were subjected to principal component analysis (PCA), and a dendrogram was constructed to demonstrate morphological relationship. According to PCA the most important variables integrated in the first principal component were mainly fruit descriptors and three traits characterizing pubescence (1 year old shoot, leaf blade, and pedicel). Cluster analysis clearly separated the accessions in Malus series. The results showed the polyphyletic origin of the section Sorbomalus. The North American apple species, M. fusca, was closely related to the species contributing to the East Asian center of the genus origin. The relict species of the European continent (M. trilobata) was grouped in near distance to the North American species belonging to section Chloromeles. The species of Sieversinae and Baccatae dominated in one main cluster indicating their close relatedness. Most of M. ×domestica accessions were grouped with M. sieversii and M. orientalis; this supports the Sievers apple as an ancestor of domesticated apple. The morphological evaluations in this study indicated a high level of phenotypic diversity in the genebank collection. These phenotypical data will increase the feasibility of prioritizing accessions based on the uniqueness of desirable morphological traits.

Erwinia amylovora is the pathogen which incites fire blight - the most devastating bacterial disease affecting pome fruit production worldwide. So far, only one functionally proven gene for resistance to this disease has been reported in Malus, even though several quantitative trait loci (QTLs) have been identified. Since some highly aggressive strains are capable of overcoming described resistances in Malus, including that of the only isolated and functionally-proven resistance gene, it is important to identify more resistance donors useful for the establishment of pyramided and durable resistance. Quantitative trait locus analyses were performed on F1 progenies derived from a cross between the Malus ×domestica cultivar, Idared, and the wild apple species M. ×arnoldiana. Progenies were artificially inoculated with two strains of E. amylovora – the moderately aggressive strain Ea222_JKI, and the highly aggressive Canadian strain, Ea3049. A QTL was identified at the distal end of linkage group (LG) 12 of M. ×arnoldiana explaining over 50% of the phenotypic variation among the progenies inoculated with Ea222_JKI. A significant QTL effect was also identified on LG12 using the phenotypic data of the progenies inoculated with Ea3049. The fire blight resistance QTL of M. ×arnoldiana (FB_Mar12) appeared to be located at a similar position as those of Evereste and/or M. floribunda clone 821 below the SSR marker Hi07f01 on LG12 of the apple genome. The results presented here suggest that this QTL could be allelic with the other reported fire blight QTLs on LG12. Nevertheless, it is imperative to perform more studies aimed at further elucidating the resistance mechanism of this wild apple species since it displays a significant resistance effect to Ea3049.

Summary Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2EA, a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2EA was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2EA of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host–pathogen system Mr5 and E. amylovora.

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

The bacteria Erwinia amylovora, the causal agent of fire blight, infects most members of the Maloideae including pear and apple. In this work the defense responses against this pathogen were monitored in two apple species grown in vitro, in the susceptible Malus domestica Borkh. cv. ‘Idared’ (later on ‘Idared’) and the resistant Malus x robusta (Carriere) Rehder var. persicifolia Rehder (Mrp). Our results indicate that the resistant plants might represent a less favorable environment for bacterial growth. At the same time, in these plants higher basic levels for some defense-related compounds such as salicylic acid or their activities such as the PAL enzyme activity can be found. In fire blight infected plants both the known pathways for SA synthesis as well as most of the phenylpropanoid genes examined were repressed due to disease, but apparently could be compensated by complex regulatory mechanisms. Not only the nature but also the quantity of defense-related compounds is likely to influence the outcome of plant–pathogen interactions.

To break the juvenile stage of apple (Malus x domestica Borkh.) we transferred the LFY gene of Arabidopsis into the genome of the apple cv. 'Pinova'. A total of five transgenic clones constitutively overexpressing the LFY gene were obtained. Approximately, 20 shoots of each clone were rooted and transferred to the glasshouse. No flowers were obtained on transgenic plants during the first 2 years of cultivation. Evaluation of the expression of possible LFY targets revealed that no transcripts could be detected for MdAP1-1 and MdAP1-2. MdTFL1 was unaffected. Based on the absence of the LFY core-binding sequence within promoter sequences of MdAP1-1 and MdAP1-2, it was concluded that LFY was not able to induce these genes. The LFY genes of apple were unaffected in transgenic plants and sequence alignments of the C-terminal amino acid sequence showed a high conservation of these proteins. A change in binding ability to DNA can therefore be excluded. Instead of early flowering, the transgenic plants showed an altered phenotype, which is similar to the columnar phenotype of the 'McIntosh Wijcik' mutant of apple. The transgenic plants showed shortened internodes and a significantly reduced length of the regrowing shoot. A negative correlation was observed between the length of the regrowing shoot and the LFY mRNA transcript level. Furthermore, the LFY transgenic apple plants showed an increased shoot diameter at node 20, which was positively correlated with the LFY mRNA transcript level. Based on our results, we assume an alternative role of LFY in apple.

The long juvenile period of trees delays the breeding of new varieties. Flowering begins within 5–10 years in most cultivated forest trees under intensive management, but can take up to 40 years in some species and environmental conditions. To accelerate the breeding process several agrotechnical and biotechnical methods have been developed. Knowledge about genes controlling flower initiation in model plants like Arabidopsis thaliana, and identification of homologous genes in trees, have led to new possibilities for early-flower induction. Overexpression of MADS-box and other floral regulatory genes resulted in early flowering in some tree species and/or varieties. However, these methods have not yet been shown to enable the production of fertile, viable or normal gametes and progeny; developmental research towards these ends is therefore of high priority. A breeding scheme has been developed to use early flowering trees for the introduction of genes from wild species that would allow several backcrosses to occur in only a few years, and to produce at the end a non-transgenic improved variety. Research to develop practical early flowering methods could lead to several new methods for breeding and biotechnology.

To shorten the juvenile stage of apple (Malus x domestica Borkh.) the BpMADS4 gene from silver birch (Betula pendula Roth.) was constitutively overexpressed in 25 transgenic apple clones. All clones were characterized by PCR, RT-PCR and Real Time PCR. Solitary flowers were produced on in vitro shoots of eight transgenic clones and most of them appeared to be morphologically normal. Twenty shoots of each clone were rooted and transferred to a glasshouse. Glasshouse plants of clones T1165, T1187 and T1190 developed flowers. Several plants of T1165 and T1187 started floral initiation within 3-4 months following transfer to the glasshouse. Primary flowers were solitary and in a terminal position on the main shoot. Lateral flower clusters, consisting of three to five individual flowers, were also found. Pollen vitality and tube germination of glasshouse-grown flowers were investigated, and there were no significant differences compared to pollen of non-transgenic control plants. Preliminary crosses using flowers of glasshouse plants resulted in small apple fruits. It would seem that this is the first report on in vitro flower induction in transgenic apple.

Cisgenic apple plants of two different cultivars were developed by transferring the Rvi6 scab resistance gene of Malus floribunda 821, using a new transformation vector based on the Flp/FRT recombinase system. Transformation experiments on seven different cultivars resulted in 22 transgenic lines for the cultivars ‘Brookfield Baigent’, ‘Mitchgla’, ‘Novajo’, and ‘Pinova’, whereby 16 lines thereof were resistant to Venturia inaequalis strain 104 (race 1). Analysis of the transgenic lines revealed Rvi6 mRNA expression levels comparable to several traditional bred Rvi6 containing cultivars and identified four transgenic lines, harboring a single T-DNA insertion, as suitable for the production of cisgenic lines. The T-DNA insertion site of these lines was determined, and lines were subject to induction of the recombinase system. Two cisgenic lines originating from the cultivars ‘Brookfield Baigent’ and ‘Pinova’ were obtained for which the exact excision of the recombinase cassette was confirmed by sequencing the previously determined T-DNA integration site. Further investigations revealed both cisgenic lines as fully resistant to V. inaequalis race 1. Rvi6 mRNA expression of the cisgenic lines and traditionally bred Rvi6 harboring cultivars was still comparable. The transformation vector developed is useable for the production of cisgenic apple plants to a certain extent.

Recently, Mfu10 - the major QTL for resistance to fire blight was reported on linkage group 10 of Malus fusca which could explain about 66 % of the phenotypic variation in a population artificially inoculated with Erwinia amylovora strain Ea222_JKI. In the meantime, it had been reported that the resistance of the fire blight QTL of Malus × robusta 5 (Mr5), which could explain 80 % of phenotypic variation is strain specific and is completely overcome by the highly virulent strain Ea3049 originating from Canada (E2002A). In contrast to Mr5, the M. fusca donor of Mfu10 is only very slightly affected by this highly virulent strain. In this short communication, we prove that this particular strain is not able to overcome the resistance of M. fusca but affects Mfu10. The same F1 progenies, derived from a cross between the resistant M. fusca and the susceptible apple cultivar, Idared, used to detect Mfu10, were used for phenotyping with Ea3049. Although the mean shoot necrosis of all progenies was 62.4 %, marker-phenotype association determined by Kruskal-Wallis analysis showed that markers on LG10 correlate significantly to resistance levels with SSR markers FR481A and FRM4 having the highest K values of 37.1 and 36.7, respectively. Interval mapping and multiple QTL mapping (MQM) performed with MAP-QTL®, showed that Mfu10 could still be detected on the same position on LG10, but only explaining 41.2 % of phenotypic variation. The implications of these results are discussed.

Rapid cycle breeding in apple is a new approach for the rapid introgression of agronomically relevant traits (e.g. disease resistances) from wild apple species into domestic apple cultivars (Malus × domestica Borkh.). This technique drastically shortens the long-lasting juvenile phase of apple. The utilization of early-flowering apple lines overexpressing the BpMADS4 gene of the European silver birch (Betula pendula Roth.) in hybridization resulted in one breeding cycle per year. Aiming for the selection of non-transgenic null segregants at the end of the breeding process, the flower-inducing transgene and the gene of interest (e.g. resistance gene) that will be introgressed by hybridization need to be located on different chromosomes. To improve the flexibility of the existing approach in apple, this study was focused on the development and characterization of eleven additional BpMADS4 overexpressing lines of four different apple cultivars. In nine lines, the flowering gene was mapped to different linkage groups. The differences in introgressed T-DNA sequences and plant genome deletions post-transformation highlighted the unique molecular character of each line. However, transgenic lines demonstrated no significant differences in flower organ development and pollen functionality compared with non-transgenic plants. Hybridization studies using pollen from the fire blight-resistant wild species accession Malus fusca MAL0045 and the apple scab-resistant cultivar 'Regia' indicated that BpMADS4 introgression had no significant effect on the breeding value of each transgenic line.

Summary The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5-virulent and Mr5-avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5-avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5-virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene-for-gene interaction in the host–pathogen relationship Mr5–E. amylovora.

TFL1 is known as a floral repressor in Arabidopsis thaliana (L.) Heynh. In apple there are two TFL1 homologs, MdTFL1-1 and MdTFL1-2. The MdTFL1-1 gene was silenced in transgenic clones expressing a hairpin gene construct of a 323 bp fragment of MdTFL1-1. The hairpin gene construct was transferred to three different apple genotypes. Of 22 transgenic clones, 21 showed a significant reduction in MdTFL1-1 mRNA expression. Precocious flowering was obtained for 20 clones, which flowered already during in vitro cultivation. Nineteen clones could successfully be transferred to the greenhouse where 18 of them flowered within a few weeks followed by the death or at least a strongly inhibited vegetative growth of the plant. Most of the transgenic flowers developed abnormally. Results obtained on greenhouse-grown plants of the transgenic clones and transgenic seedlings clearly demonstrated the major role of MdTFL1 genes in maintaining the vegetative growth as prerequisite for a perennial lifecycle. It was shown that MdTFL1 dsRNAi promotes a life history similar to annual plants. Preliminary results obtained from grafting experiments with non-transgenic scions grafted onto MdTFL1 dsRNAi transgenic rootstocks indicated that the flower-inducing signal obtained after silencing of MdTFL1 genes seems not to be graft-transmissible.

In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’ after inoculation with the fire blight bacterium, Erwinia amylovora . No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2′-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2′-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains . The most efficient compound was 3,5-dihydroxybiphenyl (MIC = 115 μg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.

Transgenic apple plants (Malus × domestica cv. ‘Holsteiner Cox’) overexpressing the Leaf Colour (Lc) gene from maize (Zea mays) exhibit strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins). Greenhouse plants investigated in this study exhibit altered phenotypes with regard to growth habit and resistance traits. Lc-transgenic plants show reduced size, transversal gravitropism of lateral shoots, reduced trichome development, and frequently reduced shoot diameter and abnormal leaf development with fused leaves. Such phenotypes seem to be in accordance with a direct or an indirect effect on polar-auxin-transport in the transgenic plants. Furthermore, leaves often develop necrotic lesions resembling hypersensitive response lesions. In tests, higher resistance against fire blight (caused by the bacterium Erwinia amylovora) and against scab (caused by the fungus Venturia inaequalis) is observed. These phenotypes are discussed with respect to the underlying altered physiology of the Lc-transgenic plants. The results are expected to be considered in apple breeding strategies.

A strong fire blight resistance QTL (Mfu10) was previously detected on linkage group 10 (LG10) of Malus fusca accession MAL0045, using several strains of the causative bacterium, Erwinia amylovora. As no strain capable of breaking the resistance of MAL0045 has been found, we hypothesized that another locus contributes to its fire blight resistance. However, none was detected with strains previously tested on the progeny. Here, an avrRpt2EA mutant strain (Ea1038) with the chromosomal S-allele deleted and complemented with the less aggressive C-allele, was used to phenotype MAL0045 × ‘Idared’ progeny. We performed phenotype-genotype analyses using the first genetic map of MAL0045, which is scarcely dense, and a recently constructed saturated map. As expected, Mfu10 was detected on LG10 with Ea1038, as was previously with other strains. Interestingly, a QTL with a logarithm of odds (LOD) thresholds of 5.5 and 2.9, significant at the genome-wide and chromosome levels, respectively, was detected with Ea1038 on LG16 (Mfu16) in a subset of 76 individuals, but only using the saturated map. Progenies carrying both Mfu10 and Mfu16 were significantly more resistant than progenies carrying only Mfu10. However, the LOD of Mfu16 diminished to 2.6 in a larger subset of individuals. We hypothesize that Mfu16 is present in the genome of MAL0045 albeit unstable in the progeny.

Fire blight caused by Erwinia amylovora is one of the most disastrous diseases in apple production. Whereas most apple cultivars are susceptible to fire blight, several wild apple species accessions like Malus ×robusta 5 (Mr5) bear significant resistance. The resistance of Mr5 is mainly inherited by a major quantitative trait locus (QTL) on linkage group 3. QTL mapping was performed after inoculation of the population 04208 (Idared × Mr5) using strains differing in their virulence to Mr5. The QTL mapping approach demonstrated that the major QTL on linkage group 3 could be confirmed after inoculation with strains non-virulent to Mr5. In contrast, the major QTL disappeared after inoculation with strains virulent to Mr5. Only after inoculation with the resistance breaking strain Ea 3049 was a minor QTL with a LOD >3 found on linkage group 3. Additionally, several minor QTLs were detected on linkage groups 5, 7, 11 and 14 of Mr5 after inoculation with virulent strains able to overcome the major resistance QTL of Mr5. Their usefulness for further breeding activities will be discussed. The strain-specific results obtained in the present study provide further evidence for the existence of gene-for-gene relationships in the host–pathogen system Mr5–E. amylovora. Of the newly discovered minor QTLs, the one detected on LG7 contributes significantly to fire blight resistance in the presence of the major QTL, independently of the strain used.