Markus Kellerhals

Vr2: a new apple scab resistance gene

Reports from several European countries of the breakdown of the Vf resistance, the most frequently used source of resistance in breeding programs against apple scab, emphasize the urgency of diversifying the basis of apple scab resistance and pyramiding different apple scab resistances with the use of their associated molecular markers. GMAL 2473 is an apple scab resistant selection thought to carry the resistance gene Vr. We report the identification by BSA of three AFLP markers and one RAPD marker associated with the GMAL 2473 resistance gene. SSRs associated with the resistance gene were found by (1) identifying the linkage group carrying the apple scab resistance and (2) testing the SSRs previously mapped in the same region. One such SSR, CH02c02a, mapped on linkage group 2, co-segregates with the resistance gene. GMAL 2473 was tested with molecular markers associated with other apple scab resistance genes, and accessions carrying known apple scab resistance genes were tested with the SSR linked to the resistance gene found in GMAL 2473. The results indicate that GMAL 2473 does not carry Vr, and that a new apple scab resistance gene, named Vr2, has been identified.

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.

Towards improvement of marker assisted selection of apple scab resistant cultivars: Venturia inaequalis virulence surveys and standardization of molecular marker alleles associated with resistance genes.

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).

A candidate gene for fire blight resistance in Malus ×robusta 5 is coding for a CC–NBS–LRR

AbstractFire blight is the most important bacterial disease in apple (Malus ×  domestica) and pear (Pyrus communis) production. Today, the causal bacterium Erwinia amylovora is present in many apple- and pear-growing areas. We investigated the natural resistance of the wild apple Malus ×  robusta 5 against E. amylovora, previously mapped to linkage group 3. With a fine-mapping approach on a population of 2,133 individuals followed by phenotyping of the recombinants from the region of interest, we developed flanking markers useful for marker-assisted selection. Open reading frames were predicted on the sequence of a BAC spanning the resistance locus. One open reading frame coded for a protein belonging to the NBS–LRR family. The in silico investigation of the structure of the candidate resistance gene against fire blight of M. ×  robusta 5, FB_MR5, led us hypothesize the presence of a coiled-coil region followed by an NBS and an LRR-like structure with the consensus ‘LxxLx[IL]xxCxxLxxL’. The function of FB_MR5 was predicted in agreement with the decoy/guard model, that FB_MR5 monitors the transcribed RIN4_MR5, a homolog of RIN4 of Arabidopsis thaliana that could interact with the previously described effector AvrRpt2EA of E. amylovora.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BackgroundThe amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North + East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe.ResultsA Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST = 0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST = 0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars.ConclusionsThe variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

European pome fruit genetic resources evaluated for disease resistance

Pome fruit genetic resources collections constitute a highly valuable resource not only for fruit breeding but also for direct use by nurseries, growers, and home gardeners. In order to use these resources efficiently and sustainably, reliable evaluation data on fruit and tree characteristics must be generated. Here we focus on pome fruit genetic resources evaluated phenotypically and genotypically for susceptibility to apple scab (Venturiainaequalis), powdery mildew (Podosphaeraleucotricha), fire blight (Erwiniaamylovora), pear rust (Gymnosporangiumsabinae) and storage diseases (e.g., Penicilliumexpansum). Examples are presented of several ongoing projects throughout Europe, with the aim to evaluate fruit genetic resources for disease susceptibility and potential use in breeding and for commercial use. The COST action 864 has fostered international cooperation in the evaluation of pome fruit genetic resources, and some of these evaluations therefore involve research groups from several of the participating countries.

Breeding Elite Lines of Apple Carrying Pyramided Homozygous Resistance Genes Against Apple Scab and Resistance Against Powdery Mildew and Fire Blight

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

Towards a marker assisted breeding for resistance against apple scab

We present the first attempts to find molecular markers (RAPDs) in the apple genome linked to the Vf gene conferring resistance to scab. The availability of genetic markers will allow selection of individuals with resistance genes in progeny. Moreover genetic markers allow us to recognise individuals with minimal linkage drag in back-crossing experiments with wild species. The feasibility of the bulk-segregant analysis to identify linked markers has been demonstrated on several occasions (Martin et al., 1991; Giovannoni et al., 1991; Michelmore et al., 1991). In this paper we report the preliminary results proving that the method could also be applied successfully in allogamous plants.

Development of SNP-based assays for disease resistance and fruit quality traits in apple (Malus × domestica Borkh.) and validation in breeding pilot studies

The development of molecular markers linked to specific traits is now routine practice, but the gap between genomics and breeding often delays their application. In the frame of the FP7 European project FruitBreedomics, apple pilot studies were designed to exploit the project’s outcomes towards the practical application of marker-assisted breeding (MAB) programs. The aim of this pilot study was to develop an outsourcing genotyping pipeline, which will provide access to the single nucleotide polymorphism (SNP) markers analysis for breeding companies without an internal DNA lab. The process from seed sowing to genotypic and phenotypic seedling selection was optimized. KASP™ (competitive allele-specific PCR) genotyping assays were developed for a number of major resistance genes for apple scab (Rvi2, Rvi4, Rvi6, and Rvi15); powdery mildew (Pl2); and rosy apple aphid (Dp-fl). In addition, KASP™ assays for the genes Md-ACS1, Md-ACO1, and Md-PG1 involved in fruit quality (firmness, texture, and storability) were also developed. The pilot study demonstrated the efficacy of the SNP-based selection strategy, especially for those programs dealing with traits not easily assessable in vivo, such as pyramided resistances and fruit quality traits.

Pyramidisieren von Schorfresistenzen in der Apfelzüchtung

ZusammenfassungQualitativ hochwertige Apfelsorten mit umfassender Krankheitsresistenz sind das wichtigste Ziel im Zuchtprogramm von Agroscope Changins-Wädenswil (ACW). Der Apfelschorf (Venturia inaequalis) ist der wichtigste Krankheitserreger im Apfelanbau. Um die Dauerhaftigkeit der Schorfresistenz zu sichern, wird eine Kombination verschiedener Resistenzgene (Pyramidisieren) angestrebt. Zudem werden Resistenzen gegen Schorf, Mehltau (Podosphaera leucotricha) und Feuerbrand (Erwinia amylovora) im gleichen Genotyp kombiniert. Die Kombination (Pyramidisieren) von Resistenzen wird am Beispiel von Kreuzungen mit zwei verschiedenen Schorfresistenzgenen (Vf und Vh2) illustriert. Kreuzungsnachkommen werden mithilfe von molekularen Markern auf Mehrfachresistenz und das Vorliegen von Resistenzgenen getestet. Die Ergebnisse des Schorfscreenings im Gewächshaus und die Markeranalysen werden miteinander verglichen und die Aufspaltung in resistente und nichtresistente Nachkommen analysiert.AbstractHigh quality disease resistant apples are the main breeding objective in the programme at Agroscope Changins-Wädenswil (ACW). Apple scab caused by the fungus Venturia inaequalis is still the most important disease in apple production. To ensure the durability of scab resistance we are combining different resistance genes (pyramiding). Moreover, we are incorporating also resistances against powdery mildew (Podosphaera leucotricha) and fire blight (Erwinia amylovora). Principles and methods of resistance breeding are outlined and pyramiding of resistances is illustrated by analysing crosses between two cultivars carrying different scab resistance genes (Vf and Vh2). Crosses are being analysed applying marker-assisted selection (MAS) to detect plants carrying multiple resistances. Phenotypic screening of seedlings for scab symptoms and MAS results are compared and the observed segregations into resistant and susceptible progenies discussed.