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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.

Premature leaf fall disease is caused by the hemibiotrophic ascomycete Diplocarpon mali (Y. Harada & Sawamura 1974) (anamorph Marssonina coronaria). Breeding for disease resistance and development of resistant cultivars could help to defeat the disease in the future. The first step in resistance breeding is to assess Malus genetic resources for resistance to M. coronaria. The aim was to proof a detached leaf assay as phenotyping procedure. Disease assessment was performed after 14 days post inoculation (dpi). Different symptoms obtained from the inoculated genotypes indicate a high level of susceptibility in Malus cultivars. In contrast, M. baccata accession MAL0419 was tolerant to M. coronaria. Future experiments will show whether the tolerance of M. baccata accession MAL0419 is inherited and if the genotype is a possible donor for resistance breeding toward M. coronaria.

Objective The proposed candidate gene underlying the Malus fusca fire blight resistance locus on chromosome 10 was previously predicted to possess 880 amino acids and 8 exons. Eight base pair (8 bp) insertion/deletion in the first exon potentially distinguished resistant genotypes from susceptible ones. This study aimed at analyzing the candidate gene sequence in another set of original resistant and susceptible progeny, characterizing the sequence in a transgenic line transformed with the candidate gene under its own native promoter, as well as deciphering the potential genomic differences between this candidate gene and its homolog in the ‘Golden Delicious’ doubled haploid genome (GDDH13). Results Sequences of amplicons of part of the candidate gene amplified in two progenies that showed resistant and susceptible fire blight phenotypes, confirmed the 8 bp insertion that distinguishes susceptible and resistant progenies. The transgenic line was positive for the candidate gene sequence, confirming a successful transfer into the background of apple cultivar ‘Pinova’, and possessed the same genomic sequence as the progeny with a resistant phenotype. Sequence analysis showed that the homolog gene on GDDH13 possesses a significant 18 bp deletion in exon 1 leading to a difference of 15 amino acid from the protein sequence of the candidate gene.

ObjectiveErwinia amylovora causes fire blight disease in Malus. A strong resistance QTL (Mfu10) was previously detected on linkage group 10 of Malus fusca accession MAL0045, using several strains of the bacterium. As no strain capable of breaking the resistance of MAL0045 has been found, it was hypothesized that a second resistance factor contributes to the fire blight resistance of MAL0045. However, to date, no minor locus has been detected with previously published strains of the bacterium. We detected a minor QTL only on a subset of a population following inoculation with strain Ea1038, which heterologously expresses an effector in a derivative of isolate Ea3049. Two genetic maps of MAL0045, one scarce, the other dense with markers, were used for QTL analyses.ResultsMfu10 was detected on LG10 with Ea1038, as was previously with Ea3049. Although no other QTLs of significant LOD was previously detected in other linkage groups with Ea3049, a QTL of significant LOD was detected on LG16 (Mfu16) after inoculation of a subset of 76 individuals with Ea1038, but only using the dense genetic map. Mfu16 improved the effect of Mfu10. However, when the number of individuals inoculated with Ea1038 was increased to 121, Mfu16 was no longer detected in the dense genetic map. We hypothesize some factors, which might be responsible for the instability of this QTL.

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.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor., Scientific Reports, 11 (1), ISSN:2045-2322

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.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor.

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.

Although, the Pacific crabapple, Malus fusca, is a hardy and disease resistant species, studies relating to the genetics of its unique traits are very limited partly due to the lack of a genetic map of this interesting wild apple. An accession of M. fusca (MAL0045) of Julius Kühn-Institut collection in Germany is highly resistant to fire blight disease, incited by different strains of the causative pathogen—Erwinia amylovora. This is the most destructive bacterial disease of Malus of which most of the domesticated apples (Malus domestica) are susceptible. Using a scarcely dense genetic map derived from a population of 134 individuals of MAL0045 × ‘Idared’, the locus (Mfu10) controlling fire blight resistance mapped on linkage group 10 (LG10) and explained up to 66% of the phenotypic variance with different strains. Although the development of robust and tightly linked molecular markers on LG10 through chromosome walking approach led to the identification of a major candidate gene, any minor effect locus remained elusive possibly due to the lack of marker density of the entire genetic map. Therefore, we have developed a dense genetic map of M. fusca using tunable genotyping-by-sequencing (tGBS) approach. Of thousands of de novo SNPs identified, 2677 were informative in M. fusca and 90.5% of these successfully mapped. In addition, integration of SNP data and microsatellite (SSR) data resulted in a final map comprising 17 LGs with 613 loci spanning 1081.35 centi Morgan (cM). This map will serve as a template for mapping using different strains of the pathogen.

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.

The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2EA) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malus × robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2EA, a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85 A. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.

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.

The apple TFL1 genes, MdTFL1-1 and MdTFL1-2, were tried to be silenced using the opposing dual-promoter gene construct 620p9U10-HSP::MdTFL1-HSP containing a 323 bp fragment of MdTFL1-1 flanked by two inverted sequences of the Gmhsp17.5-E heat-inducible promoter. This construct was used for transformation experiments with the apple cultivars ‘Gala’ and ‘Pinova’. Three (T967, T973, and T985) and two (T976 and T987) transgenic lines were obtained for ‘Pinova’ and ‘Gala’, respectively. Single heat shock treatments at 42 °C for 1 and 2 h, respectively, were applied to plants of transgenic lines and non-transformed controls grown in vitro or in the glasshouse. Heat shock treatments induced a short-term activation of the transgene construct. Silencing of MdTFL1-1 and MdTFL1-2 was detected in transgenic plants, but also in non-transformed controls, highlighting a similar heat response. The transcription of both MdTFL1 genes was repressed for 24 h post heat shock induction. In parallel, we treated shoots of glasshouse-grown plants at 42 °C for 2 h daily for the duration of 28 days. This treatment resulted in silencing of MdTFL1 in leaves of transgenic and non-transformed plants. However, repeated heat shock treatments failed to induce flower development in plants. Notwithstanding, we recorded a number of deformed leaves on transgenic shoots in comparison to non-transformed ones. This has lead to the conclusion that the transgene construct is functional in down-regulation of TFL1 homologs. The heat shock treatments demonstrated a major role of both MdTFL1 genes in plant’s heat response.

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

Fire blight is a common and very destructive disease affecting apple (Malus ×domestica) and pear (Pyrus communis) productions. Caused by enterobacterium Erwinia amylovora, its devastating economic effects ranges from losses in yield to severe damage or death of trees in the orchard, and in extreme cases loss of a whole orchard. The most effective management measure available to producers is the use of antibiotics. However, dependence on antibiotics treatment is not sustainable given risks such as the rapid selection of antibiotic-resistant Erwinia populations and environmental hazards, which have led to their application being forbidden in many countries. Therefore the planting of fire blight-resistant cultivars seems to be the most promising alternative strategy. However until now, there is no fire blight-resistant apple cultivar produced for the global market. Host resistance to fire blight in Malus is thought to be quantitatively controlled. Thus, several quantitative trait loci for resistance have been identified in some accessions of Malus spp. In this study, we explored a segregating population derived from a cross between the apple wild species Malus fusca and the Malus ×domestica cultivar ‘Idared’. F1 progenies used for mapping were artificially inoculated with E. amylovora strain Ea222 at a concentration of 109 CFU/ml in two different years. The averages of percentage lesion length (PLL) of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal-Wallis analysis, used to determine marker-phenotype association, revealed two DArT markers (971000 and 970840) with the highest significance. QTL mapping revealed a strong QTL explaining above 60% of the phenotypic variation. This is the first report of a fire blight resistance QTL in M. fusca.

To date the use of natural resistance to manage fire blight epidemics in apple orchards has been limited by the reduced availability of such traits in commercial varieties as well as the very poor fruit quality of resistant wild apple genotypes. Such natural resistance offers great environmental advantages compared to any other disease control methods i.e. less treatments with chemicals and less tractor rides. We undertook the positional cloning of the fire blight resistance gene located on the linkage group 3 of Malus ×robusta 5 (Mr5), a wild apple genotype immune to European strains of Erwinia amylovora. A single candidate gene (FB_MR5) was identified and validated by a transgenic approach, transforming the fire blight susceptible cultivar ‘Gala’. This represents an unprecedented opportunity to deploy Malus-own resistance by cisgenics, similarly to what we recently reported for the scab resistance gene Rvi6, showing that such GM product may represent an effective and sustainable approach in fire blight management. Current state of the research will be presented.

Malus × robusta 5 (Mr5) is a wild apple accession highly resistant to fire blight caused by the bacterium Erwinia amylovora. Only few strains of E. amylovora are able to overcome the resistance, including the avrRpt2EA deletion mutant ZYRKD3-1. The related wild type strain Ea1189, expressing the avrRpt2EA is not able to break resistance of Mr5. The differential transcriptome comparison of Mr5 after inoculation with these two strains gives the opportunity to analyze the host-pathogen interaction of Erwinia amylovora and Mr5 in a system differing only in one single gene: the avrRpt2EA effector gene.

Fire blight, caused by enterobacterium Erwinia amylovora, is the most important bacterial disease affecting pome fruit. It can cause devastating economic losses and is reliably controlled only by the application of antibiotics, which are banned in many European countries due to environmental and sustainability issues and consumers’ mistrust. One solution could be the utilization of fire blight resistant cultivars in apple production. In 2003, we started an approach at Dresden-Pillnitz to detect different mechanisms conferring resistance to fire blight aimed at their com-bination in new cultivars. Four segregating populations were established to map QTLs for fire blight resistance. The donors used were three wild species accessions of Malus baccata, M. fusca, M. ×robusta 5 and the Pillnitz cultivar ‘Rewena’. The susceptible parent in each case was Idared. Grafted scions of each progeny were inoculated with E. amylovora strain Ea222_JKI. Average percent lesion length (PLL) of all progenies was determined. Genetic linkage maps were established using DArT- SCAR-, SNP-, and SSR-markers. Whereas in Rewena no QTL could be determined, major QTLs were detected in M. baccata on linkage group 12, in M. fusca on linkage group 10, and in M. ×robusta 5 on linkage group 3 explaining up to around 50, 85 and 85% of the phenotypic variance, respectively. The fact that all resistance QTLs are located on different linkage groups enhances the chance that different mechanisms are acting in the donors. Additionally, trees of the Idared by M. ×robusta 5 population were planted in an orchard and flowers were inoculated for phenotyping. The QTL on linkage group 3 could be con-firmed after mapping.

Red fruit flesh is a desirable trait in apple breeding, because red-fleshed apples are a novelty and therefore considered to be more attractive to consumers and contain more health beneficial compounds. The red fruit flesh coloration is based on an increased level of cyanidin 3-galactoside, an anthocyanin whose biosynthesis is regulated by the MYB-type transcription factors MdMYB10 or MdMYB110a, respectively. A repeated segment in the MdMYB10 promoter allele R6 results in a gain-of-function mutation visible as red pigmentation of fruit skin and flesh and all vegetative tissues. Red-fleshed apple genotypes containing this R6 allele belong to the type 1 red-fleshed apple, which is known to be linked to some negative traits like astringent taste and internal flesh browning disorder. In type 2 red-fleshed apples the fruit flesh coloration is not inevitably linked with skin and leaf color. This red-fleshed apple phenotype, which is a result of increased expression of MdMYB110a, seems to be more useful for breeding, but it can be found rather seldom. In the present study 357 Malus accessions of the German Malus Germplasm Collection were evaluated for red fruit flesh coloration and the presence of the MdMYB10 R1 (not mutated) and R6 promoter alleles. Among them a total of 40 accessions were identified which contain the R6 allele. 37 accessions showed a red coloration of the fruit flesh. All these accessions belong to type 1 red-fleshed apple. No type 2 red-fleshed apple could be found. Three accessions with R6 allele had non-red-fleshed apples. 312 other non-red-fleshed accessions contained only the R1 allele. Five non-red-fleshed accessions contained a new promoter allele with an unexpected size of ~1 kbp. Sequencing of this allele detected the insertion of a non-autonomous apple transposon.

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.

Few complete genes belonging to the receptor-like protein class of plant resistance (R) genes (called HcrVf genes in Malus) have been cloned from apple cultivars. To date, the HcrVf2 gene from the Rvi6 locus of ‘Florina’, a derivative of Malus × floribunda 821, is the only cloned apple scab R gene with a proven function. The breakdown of the Rvi6 scab resistance in several apple growing regions has forced the search for new resistance sources for R gene pyramiding through traditional and biotechnological breeding. Marker-assisted breeding is aimed at the selection of the desired R gene combinations but might be extended for monitoring putative risks of resistance breakdown in potential scab R gene donors. Here we report on a marker-based screen of Rvi6 homologues supplemented by a polymerase chain reaction (PCR)-based full-length cloning of HcrVf paralogs. Known Rvi6 markers were analysed in a sub-set of accessions selected by a preceding SSR-based genetic relationship analysis from a large Malus species germplasm collection, which has been evaluated for scab resistance in an unsprayed orchard for a period of 3 years. The Rvi6 breakdown in several M. × floribunda accessions was confirmed, and several other Malus species putatively related to M. × floribunda were also infected by scab. The selected sub-cluster consisting of 40 accessions, including all M. × floribunda, two Malus × micromalus and two Malus baccata accessions, was screened for Rvi6 markers CH-Vf1-SSR and AL07-SCAR and for the presence of HcrVf2 by using gene-specific primers. The two M. × micromalus accessions, which proved to be identical genotypes, were found to be closely related to M. × floribunda. They also displayed the Rvi6 markers and could be infected by race (5,6,7) scab isolate Vi158. To verify the assumed existence of the HcrVf2 gene in M. × micromalus, a PCR-based cloning method was used to clone full-length HcrVf paralogs from this species and additionally from a scab-susceptible M. baccata genotype also showing the Rvi6 markers. The M. × micromalus gene MAM31 was identified as an identical copy of HcrVf2. Another HcrVf-like gene (MAM6) newly cloned from M. × micromalus showed 95 % similarity to HcrVf2. MAM6 was chosen for the development of a gene-specific PCR marker, which was analysed in the selected apple group and additionally mapped in an apple progeny derived from a cross with M. × micromalus. The cloning method described in this paper might be used in future to mine for more HcrVf gene variants to develop highly specific markers for R gene deployment in traditional breeding and to use cloned genes for gene transfer and functional studies.

Apple Malus × domestica Borkh. has a long period of juvenility during which plants are unable to produce flowers and fruits. Flowering is only possible on trees which have passed the transition from juvenility to the adult stage. Several genes have been identified which control this switch and promote or delay flowering in different plant species. FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) are two flowering genes of Arabidopsis which are similar, but act antagonistically. FT and TFL1 homologous genes are present in the apple genome, but little is known about their function. Therefore we produced transgenic apple lines which over-expressed the MdFT2 gene under the control of the constitutive CaMV 35S promoter. Transgenic shoots expressing the MdFT2 gene driven by the CaMV 35S promoter flowered in vitro. To study the function of MdTFL1 we silenced the gene by expressing an MdTFL1 specific hairpin under the control of the CaMV 35S promoter. Transgenic plants of different apple cultivars flowered in vitro and later in the greenhouse. Transgenic flowers were evaluated for phenotypic differences and tested on their usability for crossbred breeding programs.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; 'Antonovka' has long been recognised as a major source of scab (Venturia inaequalis) resistance useful for apple breeding worldwide. Both major gene resistances in the form of the Rvi10 and Rvi17 and quantitative resistance, collectively identified as VA, have been identified in different accessions of 'Antonovka'. Most of the 'Antonovka' scab resistance used in apple-breeding programmes around the world can be traced back to Schmidt 'Antonovka' and predominantly its B VIII progenies 33,25 (PI 172623), 34,6 (PI 172633), 33,8 (PI 172612) and 34,5 (PI 172632). Using genetic profile reconstruction, we have identified "common 'Antonovka' " as the progenitor of the B VIII family, which is consistent with it having been a commercial cultivar in Poland and the single source of scab resistance used by Dr. Martin Schmidt. The major 'Antonovka' scab resistance genes mapped to date are located either very close to Rvi6, or about 20-25 cM above it, but their identities need further elucidation. The presence of the 139 bp allele of the CH-Vf1 microsatellite marker known to be associated with Rvi17 (Va1) in most of the 'Antonovka' germplasm used in breeding suggests that it plays a central role in the resistance. The nature and the genetic relationships of the scab resistance in these accessions as well as a number of apple cultivars derived from 'Antonovka', such as, 'Freedom', 'Burgundy' and 'Angold', are discussed. The parentage of 'Reglindis' is unclear, but the cultivar commercialised as 'Reglindis' was confirmed to be an Rvi6 cultivar.

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.

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.

Breeding for fire blight resistance is a major goal of nearly all apple breeding programs worldwide. Resources for resistance can be found in Malus wild species accession such as Malus ×robusta 5 (Mr5) which carries the FB_MR5 CC-NBS-LRR fire blight resistance gene. To study the occurrence of FB_MR5 in other genetic resources of Malus, a total of 394 wild species accessions of three international germplasm collections were screened using the markers CH03e03 and FEM18 that flank the FB_MR5 locus of Mr5. Five accessions exhibited the allele sizes linked to FB_MR5 for both markers. All five accessions belong to the species M. ×robusta (Carriere) Rehder. In addition, several accessions of other closely related Malus wild species amplified the 224 bp fragment linked to FEM18 and a slightly larger fragment for CH03e03. The presence and expression of FB_MR5 and homologs in M. ×robusta and four more accessions were further tested using gene-specific primers. Fragments of identical size could be detected in six different genotypes of M. ×robusta. Sequence analysis revealed the presence of alleles of FB_MR5 in the accessions ‘Korea’ and ‘Leucocarpa’. Homologs of FB_MR5 were found in different accessions of Malus prunifolia and Malus baccata, two Malus species closely related to M. ×robusta. Their functionality is suggested by results obtained by artificial shoot inoculation with the pathogen. Based on the results using the simple sequence repeat (SSR) markers CH03e03 and FEM18, the gene-specific primers and the analysis of the ‘Golden Delicious’ whole genome sequence, we assume that FB_MR5 is a unique gene which most likely evolved from M. baccata as the common ancestor of M. ×robusta and M. prunifolia.

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.

International audience; Key message: Proof of concept of Bayesian integrated QTL analyses across pedigree-related families from breeding programs of an outbreeding species. Results include QTL confidence intervals, individuals' genotype probabilities and genomic breeding values. Abstract: Bayesian QTL linkage mapping approaches offer the flexibility to study multiple full sib families with known pedigrees simultaneously. Such a joint analysis increases the probability of detecting these quantitative trait loci (QTL) and provide insight of the magnitude of QTL across different genetic backgrounds. Here, we present an improved Bayesian multi-QTL pedigree-based approach on an outcrossing species using progenies with different (complex) genetic relationships. Different modeling assumptions were studied in the QTL analyses, i.e., the a priori expected number of QTL varied and polygenic effects were considered. The inferences include number of QTL, additive QTL effect sizes and supporting credible intervals, posterior probabilities of QTL genotypes for all individuals in the dataset, and QTL-based as well as genome-wide breeding values. All these features have been implemented in the FlexQTL (TM) software. We analyzed fruit firmness in a large apple dataset that comprised 1,347 individuals forming 27 full sib families and their known ancestral pedigrees, with genotypes for 87 SSR markers on 17 chromosomes. We report strong or positive evidence for 14 QTL for fruit firmness on eight chromosomes, validating our approach as several of these QTL were reported previously, though dispersed over a series of studies based on single mapping populations. Interpretation of linked QTL was possible via individuals' QTL genotypes. The correlation between the genomic breeding values and phenotypes was on average 90 %, but varied with the number of detected QTL in a family. The detailed posterior knowledge on QTL of potential parents is critical for the efficiency of marker-assisted breeding.

Tree Genetics & Genomes, 9 (1), ISSN:1614-2950, ISSN:1614-2942

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.

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 avrRpt2(EA) , a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2(EA) 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 avrRpt2(EA) 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.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Background: Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus 'Robusta 5'. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results: When several defined E. amylovora strains were used to inoculate three progenies from international breeding programs, all with 'Robusta 5' as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand 'Malling 9' X 'Robusta 5' population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein (MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German 'Idared' X 'Robusta 5' population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene (HSP90). In the US 'Otawa3' X 'Robusta5' population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. Conclusions: The results suggest that the upper region of 'Robusta5' linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

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.

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.