Genetic Variability of Phyllosticta ampelicida, the Agent of Black Rot Disease of Grapevine.

Phyllosticta ampelicida causes black rot disease of Vitis spp. Genetic homogeneity of pathogen populations was investigated by analyzing the number of haplotypes present in infected samples from Europe and America. The fungus was identified from an analysis of the internal transcribed spacer (ITS)1-ITS2 region, and partial sequences of ß-tubulin and calmodulin genes. The analysis of nuclear microsatellites applied to strains from Vitis spp. confirmed the existence of a high degree of genetic variability in the fungal populations, revealed four subpopulations, and showed that strains from America are distinct from the European ones. Furthermore, the results obtained by landscape genetics showed that there were different introductions of the pathogen in the main vine areas of Europe, confirming what was observed in the first reports of the disease. The genetic variability of the fungus revealed by this study confirms the ability to generate new haplotypes by sexual reproduction. The difference found between the European populations and the American one confirms that the pathogen originated from America.

Incomplete Infection of Secondarily Infected Potato Plants - an Environment Dependent Underestimated Mechanism in Plant Virology.

The common assumption in potato virus epidemiology is that all daughter tubers produced by plants coming from infected mother tubers (secondary infection) will become infected via systemic translocation of the virus during growth. We hypothesize that depending on the prevalent environmental conditions, only a portion of the daughter tubers of a plant that is secondarily infected by viruses may become infected. To test this hypothesis experimental data from standardized field experiments were produced in three contrasting environments at 112, 3280, and 4000 m a.s.l. in Peru during two growing seasons. In these experiments, the percentage of infected daughter tubers produced by seed tubers that were infected with either potato potexvirus X (PVX), potato Andean mottle comovirus (APMoV), potato potyvirus Y (PVY) (jointly infected with PVX) or potato leafroll luteovirus (PLRV) was determined. Incomplete autoinfection was found in all cases, as the percentage of virus infected daughter tubers harvested from secondarily infected plants was invariably less than 100%, with the lowest percentage of infection being 30%. Changing the growing site to higher altitudes decreased autoinfection for all viruses. Therefore, the assumption of complete autoinfection of secondarily infected plants were rejected, while the hypothesis of environmentally dependent incomplete autoinfection was accepted. The findings help explain the occurrence of traditional seed management practices in the Andes and may help to develop locally adapted seed systems in environments of the world that have no steady access to healthy seed tubers coming from a formally certified seed system. The results obtained almost three decades ago are discussed in light of most recent knowledge on epigenetic regulation of host plant - virus interactions which allow for speculating about the underlying biological principles of the incomplete autoinfection. A research roadmap is proposed for achieving explicit experimental proof for the epigenetic regulation of incomplete autoinfection in the pathosystem under study.

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars.

MAIN CONCLUSION: The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. ß-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

Development of the First Cisgenic Apple with Increased Resistance to Fire Blight.

The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar 'Gala Galaxy' using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.

Cisgenic apple trees; development, characterization, and performance.

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Transcriptional Reprogramming of the Mycoparasitic Fungus Ampelomyces quisqualis During the Powdery Mildew Host-Induced Germination.

Ampelomyces quisqualis is a mycoparasite of a diverse range of phytopathogenic fungi associated with the powdery mildew disease. Among them are several Erysiphaceae species with great economic impact on high-value crops such as grape. Due to its ability to parasitize and prevent the spread of powdery mildews, A. quisqualis has received considerable attention for its biocontrol potential. However, and in sharp contrast to the extensively studied biocontrol species belonging to the genus Trichoderma, little is known about the biology of A. quisqualis at the molecular and genetic levels. We present the first genome-wide transcription profiling in A. quisqualis during host-induced germination. A total of 1,536 putative genes showed significant changes in transcription during the germination of A. quisqualis. This finding denotes an extensive transcriptional reprogramming of A. quisqualis induced by the presence of the host. Several upregulated genes were predicted to encode for putative mycoparasitism-related proteins such as secreted proteases, virulence factors, and proteins related to toxin biosynthesis. Our data provide the most comprehensive sequence resource currently available for A. quisqualis in addition to offering valuable insights into the biology of A. quisqualis and its mycoparasitic lifestyle. Eventually, this may improve the biocontrol capacity of this mycoparasite.

Engineering fire blight resistance into the apple cultivar 'Gala' using the FB_MR5 CC-NBS-LRR resistance gene of Malus × robusta 5.

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.

Moderate warming in microcosm experiment does not affect microbial communities in temperate vineyard soils.

Changes in the soil microbial community structure can lead to dramatic changes in the soil ecosystem. Temperature, which is projected to increase with climate change, is commonly assumed to affect microbial communities, but its effects on agricultural soils are not fully understood. We collected soil samples from six vineyards characterised by a difference of about 2 °C in daily soil temperature over the year and simulated in a microcosm experiment different temperature regimes over a period of 1 year: seasonal fluctuations in soil temperature based on the average daily soil temperature measured in the field; soil temperature warming (2 °C above the normal seasonal temperatures); and constant temperatures normally registered in these temperate soils in winter (3 °C) and in summer (20 °C). Changes in the soil bacterial and fungal community structures were analysed by automated ribosomal intergenic spacer analysis (ARISA). We did not find any effect of warming on soil bacterial and fungal communities, while stable temperatures affected the fungal more than the bacterial communities, although this effect was soil dependent. The soil bacterial community exhibited soil-dependent seasonal fluctuations, while the fungal community was mainly stable. Each soil harbours different microbial communities that respond differently to seasonal temperature fluctuations; therefore, any generalization regarding the effect of climate change on soil communities should be made carefully.

Molecular characterization of cisgenic lines of apple 'Gala' carrying the Rvi6 scab resistance gene.

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple 'Gala' were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab-resistant apple 'Florina' (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07- and 0.002-fold compared with the natural expression of 'Florina', this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab-susceptible 'Gala'. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site-such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.

RNA-Seq analysis reveals candidate genes for ontogenic resistance in Malus-Venturia pathosystem.

Ontogenic scab resistance in apple leaves and fruits is a horizontal resistance against the plant pathogen Venturia inaequalis and is expressed as a decrease in disease symptoms and incidence with the ageing of the leaves. Several studies at the biochemical level tried to unveil the nature of this resistance; however, no conclusive results were reported. We decided therefore to investigate the genetic origin of this phenomenon by performing a full quantitative transcriptome sequencing and comparison of young (susceptible) and old (ontogenic resistant) leaves, infected or not with the pathogen. Two time points at 72 and 96 hours post-inoculation were chosen for RNA sampling and sequencing. Comparison between the different conditions (young and old leaves, inoculated or not) should allow the identification of differentially expressed genes which may represent different induced plant defence reactions leading to ontogenic resistance or may be the cause of a constitutive (uninoculated with the pathogen) shift toward resistance in old leaves. Differentially expressed genes were then characterised for their function by homology to A. thaliana and other plant genes, particularly looking for genes involved in pathways already suspected of appertaining to ontogenic resistance in apple or other hosts, or to plant defence mechanisms in general. IN THIS WORK, FIVE CANDIDATE GENES PUTATIVELY INVOLVED IN THE ONTOGENIC RESISTANCE OF APPLE WERE IDENTIFIED: a gene encoding an "enhanced disease susceptibility 1 protein" was found to be down-regulated in both uninoculated and inoculated old leaves at 96 hpi, while the other four genes encoding proteins (metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3) were found to be constitutively up-regulated in inoculated and uninoculated old leaves. The modulation of the five candidate genes has been validated using the real-time quantitative PCR. Thus, ontogenic resistance may be the result of the corresponding up- and down-regulation of these genes.

QTL validation and stability for volatile organic compounds (VOCs) in apple.

The aroma trait in apple is a key factor for fruit quality strongly affecting the consumer appreciation, and its detection and analysis is often an extremely laborious and time consuming procedure. Molecular markers associated to this trait can to date represent a valuable selection tool to overcome these limitations. QTL mapping is the first step in the process of targeting valuable molecular markers to be employed in marker-assisted breeding programmes (MAB). However, a validation step is usually required before a newly identified molecular marker can be implemented in marker-assisted selection. In this work the position of a set of QTLs associated to volatile organic compounds (VOCs) was confirmed and validated in three different environments in Switzerland, namely Wädenswil, Conthey and Cadenazzo, where the progeny 'Fiesta×Discovery' was replicated. For both QTL identification and validation, the phenotypic data were represented by VOCs produced by mature apple fruit and assessed with a Proton Transfer Reaction-Mass Spectrometer (PTR-MS) instrument. The QTL-VOC combined analysis performed among these three locations validated the presence of important QTLs in three specific genomic regions, two located in the linkage group 2 and one in linkage group 15, respectively, for compounds related to esters (m/z 43, 61 and 131) and to the hormone ethylene (m/z 28). The QTL set presented here confirmed that in apple some compounds are highly genetically regulated and stable across environments.

Abiotic stresses affect Trichoderma harzianum T39-induced resistance to downy mildew in grapevine.

Enhancement of plant defense through the application of resistance inducers seems a promising alternative to chemical fungicides for controlling crop diseases but the efficacy can be affected by abiotic factors in the field. Plants respond to abiotic stresses with hormonal signals that may interfere with the mechanisms of induced systemic resistance (ISR) to pathogens. In this study, we exposed grapevines to heat, drought, or both to investigate the effects of abiotic stresses on grapevine resistance induced by Trichoderma harzianum T39 (T39) to downy mildew. Whereas the efficacy of T39-induced resistance was not affected by exposure to heat or drought, it was significantly reduced by combined abiotic stresses. Decrease of leaf water potential and upregulation of heat-stress markers confirmed that plants reacted to abiotic stresses. Basal expression of defense-related genes and their upregulation during T39-induced resistance were attenuated by abiotic stresses, in agreement with the reduced efficacy of T39. The evidence reported here suggests that exposure of crops to abiotic stress should be carefully considered to optimize the use of resistance inducers, especially in view of future global climate changes. Expression analysis of ISR marker genes could be helpful to identify when plants are responding to abiotic stresses, in order to optimize treatments with resistance inducers in field.

Microbial community structure in vineyard soils across altitudinal gradients and in different seasons.

Microbial communities living in nine vineyards distributed over three altitudinal transects were studied over 2 years. Fungal and bacterial community dynamics were explored using automated ribosomal intergenic spacer analysis (ARISA) and by determining bacterial cells and fungal colony-forming units (CFUs). Moreover, extensive chemical and physical analyses of the soils were carried out. Multivariate analyses demonstrated that bacterial and fungal communities are affected by altitude, which acts as a complex physicochemical gradient. In fact, soil moisture, Al, Mg, Mn and clay content are changing with altitude and influencing the bacterial genetic structure, while in the case of fungi, soil moisture, B and clay content are found to be the main drivers of the community. Moreover, other exchangeable cations and heavy metals, not correlating with altitude, are involved in the ordination of the sites, especially Cu. Qualitative ARISA revealed the presence of a stable core microbiome of operational taxonomic units (OTUs) within each transect, which ranged between 57% and 68% of total OTUs in the case of fungi and between 63% and 72% for bacteria. No seasonal effect on the composition of microbial communities was found, demonstrating that bacterial and fungal communities in vineyards are mostly stable over the considered seasons.

Comparative RNA-seq analysis of early-infected peach leaves by the invasive phytopathogen Xanthomonas arboricola pv. pruni.

Xanthomonas arboricola pv. pruni is a quarantine bacterial pathogen that threatens peach production by causing necrotic spots on leaves and fruits, thus with the potential of severely reducing yields. The current understanding of the host plant defense responses to the pathogen is very limited. Using whole transcriptome sequencing, differential gene expression was analyzed at two time points, 2 h and 12 h post inoculation (hpi), by comparing the inoculated samples to their respective controls. On the total of 19,781 known peach genes that were expressed in all time points and conditions, 34 and 263 were differentially expressed at 2 and 12 hpi, respectively. Of those, 82% and 40% were up-regulated, respectively; and 18% and 60% were down-regulated, respectively. The functional annotation based on gene ontology (GO) analysis highlighted that genes involved in metabolic process and response to stress were particularly represented at 2 hpi whereas at 12 hpi cellular and metabolic processes were the categories with the highest number of genes differentially expressed. Of particular interest among the differentially expressed genes identified were several pathogen-associated molecular pattern (PAMP) receptors, disease resistance genes including several RPM1-like and pathogenesis related thaumatin encoding genes. Other genes involved in photosynthesis, in cell wall reorganization, in hormone signaling pathways or encoding cytochrome were also differentially expressed. In addition, novel transcripts were identified, providing another basis for further characterization of plant defense-related genes. Overall, this study gives a first insight of the peach defense mechanisms during the very early stages of infection with a bacterial disease in the case of a compatible interaction.

Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification.

Guignardia bidwellii is the etiological agent of grape black rot, a disease affecting Vitis and other Vitaceae that can cause heavy crop losses in vineyards. Its identification is based mainly on morphological characters and the symptoms on plants but, due to their variability, they may be difficult to interpret to reliably distinguish the pathogen to species. To date, despite the economic importance of G. bidwellii, no molecular investigations have been carried out on Vitis isolates and few sequence data are available for cultures derived from ornamental host plants. We analyzed samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins by morphological, molecular and proteomic techniques, including ITS1-ITS2 regions and calmodulin gene sequencing, as well as matrix-assisted laser desorption/ionization analysis by time-of-flight mass spectrometry (MALDI-TOF MS). This polyphasic approach allowed assessing the phylogenetic relationships among the different isolates and suggested the existence of two distinct species. The advantages of a polyphasic approach for the identification of G. bidwellii are highlighted.

Quantification of Venturia inaequalis Growth in Malus × domestica with Quantitative Real-Time Polymerase Chain Reaction.

A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus × domestica. The method is based on dual-labeled hybridization probes, allowing simultaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible 'Gala' was 118 times higher than in resistant 'Florina' and 'Discovery' while intermediate values were found with the intermediate susceptible 'Milwa'. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.

The development of a cisgenic apple plant.

Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g. antibiotic or herbicide resistance) in the final product and the fact that the inserted gene(s) derive from organisms sexually compatible with the target crop should rise less environmental concerns and increase consumer's acceptance. Here we report the generation of a cisgenic apple plant by inserting the endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated transformation combined with a positive and negative selection system and a chemically inducible recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for detailed investigation and were shown to carry a single T-DNA insertion and express the target gene HcrVf2. This is the first report of the generation of a true cisgenic plant.

Identification of serine/threonine kinase and nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the fire blight resistance quantitative trait locus of apple cultivar 'Evereste'.

Fire blight is the most destructive bacterial disease affecting apple (Malus×domestica) worldwide. So far, no resistance gene against fire blight has been characterized in apple, despite several resistance regions having been identified. A highly efficacious resistance quantitative trait locus (QTL) was localized on linkage group 12 (LG12) of the ornamental cultivar 'Evereste'. A marker previously reported to be closely linked to this resistance was used to perform a chromosome landing. A bacterial artificial chromosome (BAC) clone of 189 kb carrying the fire blight resistance QTL was isolated and sequenced. New microsatellite markers were developed, and the genomic region containing the resistance locus was limited to 78 kb. A cluster of eight genes with homologies to already known resistance gene structures to bacterial diseases was identified and the corresponding gene transcription was verified. From this cluster, two genes were recognized in silico as the two most probable fire blight resistance genes showing homology with the Pto/Prf complex in tomato.

Genetic mapping of 14 avirulence genes in an EU-B04 × 1639 progeny of Venturia inaequalis.

Durable resistance to apple scab (Venturia inaequalis (Cke) Wint; anamorph Spilocaea pomi Fries) is one of the major goals of apple (Malus) breeding programs. Since current scab resistance breeding is heavily reliant on genes with gene-for-gene relationships, a good understanding of the genetic basis of host-pathogen interactions needs to be developed for this strategy to be successful. While the genomic organization of apple scab resistance genes has been studied extensively, little is known about the avirulence genes in the pathogen. The progeny of a cross of European V. inaequalis race (1) isolate EU-B04 and race (1,2,8,9) isolate 1639 was used to generate a genetic map based on microsatellite and AFLP markers, and investigated for inheritance of avirulence traits on 20 Malus accessions representing 17 scab resistance genes. The accessions comprised scab differential hosts (0), (1), (2), (8), and (9), and hosts carrying known as well as not previously reported secondary resistance genes, including some identified in crosses that have resistant accessions 'Geneva', 'Dolgo', Malus baccata jackii, M. micromalus, or 'Antonovka' in their pedigree. The latter genes appear to be narrow spectrum genes that showed gene-for-gene relationships as a segregation ratio of Avr:avr=1:1 was observed on 12 accessions, while a ratio of 3:1 was observed on five accessions and a ratio of 7:1 on one host. All progenies were shown to be pathogenic, as all of them were able to infect hosts (0) and (1). A genetic map consisting of 15 major linkage groups (LGs) and spanning 972cM was generated with the aid of 156 markers. The map position of 12 avirulence traits was determined: eight avirulence genes mapped into two separate clusters (1: AvrVdg2, AvrVv1, AvrVu1, AvrVrjrd; and 2: AvrVu2, AvrVh3.2, AvrVs1, AvrVu4), while four avirulence genes (AvrRvi8, AvrVv2, AvrVt57 and AvrVsv) mapped to different LGs. AvrRvi2 and AvrRvi9 also are genetically linked, but showed an interaction with AvrRvi8, the nature of which is unclear. While AvrRvi8 segregated at 1:1 ratio, the other two Avrs segregated at 3:1 ratios. However, all progeny avirulent on hosts (2) and (9) were also avirulent on host (8) and further research is required to determine the avirulence gene relationships. A further two independently segregating loci, AvrRvi1 and AvrRvi6, identified in previous studies, were mapped by inference based on their known linkage to SSR markers. The clustering of avirulence genes in V. inaequalis reflecting the clustering of resistance genes in Malus suggests this pathosystem is a classical example of an "arms race" between host and pathogen. This also seems to apply to the narrow spectrum scab resistance genes, which may imply a larger role in plant defense for these genes than has been assumed to date.

The Rvi15 (Vr2) apple scab resistance locus contains three TIR-NBS-LRR genes.

Scab caused by the pathogen Venturia inaequalis is considered the most important fungal disease of cultivated apple (Malus x domestica Borkh.). In all, 16 monogenic resistances against scab have been found in different Malus spp. and some of them are currently used in apple breeding for scab-resistant cultivars. However, the self incompatibility and the long generation time of Malus spp. together with the high standards of fruit quality demanded from the fresh market render the breeding of high-quality cultivars in apple a long and expensive task. Therefore, the cloning of disease resistance genes and the use of the cloned genes for the transformation of high-quality apple cultivars could be an approach to solve these drawbacks. We report the construction of a bacterial artificial chromosome (BAC) contig spanning the Rvi15 (Vr2) apple scab resistance locus using two GMAL 2473 BAC libraries. A single BAC clone of the contig was sufficient to span the resistance locus. The BAC clone was completely sequenced, allowing identification of a sequence of 48.6 kb going from the two closest markers (ARGH17 and 77G20RP) bracketing Rvi15 (Vr2). Analysis of the 48.6-kb sequence revealed the presence of three putative genes characterized by a Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure. All three genes were found to be transcribed.