Fire blight resistance of the wild apple species Malus fusca

Erwinia amylovora is the pathogen responsible for inciting fire blight - the most dreaded bacterial disease of apple (Malus × domestica) and other members of the Rosaceae family. The disease is very destructive as is difficult to control. Even though fire blight was first observed over two centuries ago in America, no sustainable control measure is known till date. Disease management practices such as pruning of affected tissues as well as the application of copper and antibiotics, for example, streptomycin, are to minimize the population of E. amylovora in an orchard. However, the use of streptomycin is not allowed in many European countries as a consequence of its environmental risks and the issue of raising antibiotic resistant E. amylovora populations. Therefore, natural resistance is thought to be the most sustainable approach to manage fire blight. Genetic resistance has been investigated in Malus leading to the detection of several quantitative trait loci (QTLs) in apple cultivars and apple wild species accessions. Nevertheless, only one functionally characterized fire blight resistance gene has been isolated till date. This situation, coupled with the proof that resistance is strain specific, reinforces the need to detect more donors that could be used to establish durable resistance against fire blight. For this reason, the works described in this thesis aimed at investigating fire blight resistance in another apple wild species – Malus fusca. Different accessions of M. fusca were phenotyped in JKI, Dresden, Germany to ascertain the accession with a high resistance level. Accession MAL0045, found as having a very high resistance level was then crossed with the very susceptible apple cultivar ‘Idared’ to establish a segregating F1 population of 134 individuals. To facilitate the development of a genetic map of M. fusca, molecular markers such as DArT (Diversity Arrays Technology), SNPs (Single Nucleotide Polymorphisms) and SSRs (Simple Sequence Repeats) were developed, sourced, tested and polymorphic ones applied to the 134 individuals and then mapped. The phenotypic and genotypic data were employed for QTL analyses which resulted in the identification of a major quantitative trait locus which is located on linkage group 10 (LG10) of the apple genome and could explain about 66 % of the phenotypic variation; the second highest effect of all QTLs previously detected in Malus. Furthermore, this thesis also describes the stability and validation of the M. fusca fire blight resistance locus (Mfu10) after another phenotypic evaluation of the F1 population with a highly virulent E. amylovora isolate Ea3049 originating from Canada. Moreover, the fine mapping of the resistance region was undertaken via chromosome walking approach with the development of closely linked SSR markers suitable for marker assisted selection (MAS). For this purpose, the population was substantially increased to 1,336 individuals from an additional cross of M. fusca × ‘Idared’ and a reciprocal cross of ‘Idared’ × M. fusca. Genotyping of the whole population allowed for the identification of individuals showing recombination events within the interval of the QTL region. Phenotyping of recombinant individuals ensured that the exact position of the QTL was well defined. The first steps towards uncovering the underlying gene(s) responsible for the resistance of fire blight in M. fusca have been achieved with the development and screening of a M. fusca bacterial artificial chromosome (BAC) library with SSR markers closely linked to Mfu10 and the identification of some BAC clones in the QTL interval. This is the first report of a major quantitative trait locus for resistance to fire blight in this wild relative of apple. The implications of the results obtained in these research works in respect to breeding for resistance against the very destructive fire blight disease of Malus are discussed extensively.Erwinia amylovora is the pathogen responsible for inciting fire blight - the most dreaded bacterial disease of apple (Malus × domestica) and other members of the Rosaceae family. The disease is very destructive as is difficult to control. Even though fire blight was first observed over two centuries ago in America, no sustainable control measure is known till date. Disease management practices such as pruning of affected tissues as well as the application of copper and antibiotics, for example, streptomycin, are to minimize the population of E. amylovora in an orchard. However, the use of streptomycin is not allowed in many European countries as a consequence of its environmental risks and the issue of raising antibiotic resistant E. amylovora populations. Therefore, natural resistance is thought to be the most sustainable approach to manage fire blight. Genetic resistance has been investigated in Malus leading to the detection of several quantitative trait loci (QTLs) in apple cultivars and apple wild species accessions. Nevertheless, only one functionally characterized fire blight resistance gene has been isolated till date. This situation, coupled with the proof that resistance is strain specific, reinforces the need to detect more donors that could be used to establish durable resistance against fire blight. For this reason, the works described in this thesis aimed at investigating fire blight resistance in another apple wild species – Malus fusca. Different accessions of M. fusca were phenotyped in JKI, Dresden, Germany to ascertain the accession with a high resistance level. Accession MAL0045, found as having a very high resistance level was then crossed with the very susceptible apple cultivar ‘Idared’ to establish a segregating F1 population of 134 individuals. To facilitate the development of a genetic map of M. fusca, molecular markers such as DArT (Diversity Arrays Technology), SNPs (Single Nucleotide Polymorphisms) and SSRs (Simple Sequence Repeats) were developed, sourced, tested and polymorphic ones applied to the 134 individuals and then mapped. The phenotypic and genotypic data were employed for QTL analyses which resulted in the identification of a major quantitative trait locus which is located on linkage group 10 (LG10) of the apple genome and could explain about 66 % of the phenotypic variation; the second highest effect of all QTLs previously detected in Malus. Furthermore, this thesis also describes the stability and validation of the M. fusca fire blight resistance locus (Mfu10) after another phenotypic evaluation of the F1 population with a highly virulent E. amylovora isolate Ea3049 originating from Canada. Moreover, the fine mapping of the resistance region was undertaken via chromosome walking approach with the development of closely linked SSR markers suitable for marker assisted selection (MAS). For this purpose, the population was substantially increased to 1,336 individuals from an additional cross of M. fusca × ‘Idared’ and a reciprocal cross of ‘Idared’ × M. fusca. Genotyping of the whole population allowed for the identification of individuals showing recombination events within the interval of the QTL region. Phenotyping of recombinant individuals ensured that the exact position of the QTL was well defined. The first steps towards uncovering the underlying gene(s) responsible for the resistance of fire blight in M. fusca have been achieved with the development and screening of a M. fusca bacterial artificial chromosome (BAC) library with SSR markers closely linked to Mfu10 and the identification of some BAC clones in the QTL interval. This is the first report of a major quantitative trait locus for resistance to fire blight in this wild relative of apple. The implications of the results obtained in these research works in respect to breeding for resistance against the very destructive fire blight disease of Malus are discussed extensively

Malus Hosts–Erwinia amylovora Interactions: Strain Pathogenicity and Resistance Mechanisms

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 impact of Mendelian genetics on the breeding of apple and sweet cherry

Mendels Einfluss auf die moderne Obstzüchtung ist unverkennbar. Die von ihm aufgestellten Vererbungsregeln haben es Züchtern ermöglicht, Kreuzungsprogramme gezielt zu designen, Vorhersagen über den möglichen Erfolg zu treffen und die Selektion von Nachkommen mit verbesserten Merkmalskombinationen mithilfe molekulargenetischer Marker kostengünstig und effizient zu gestalten. Am Beispiel des Apfels und der Süßkirsche als Vertreter für Kern- und Steinobst werden ausgewählte mendelnde Merkmale vorgestellt, die von besonderer Bedeutung für die Züchtung sind. Darüber hinaus wird der derzeitige Stand an molekularen Markern und genetischen Karten bei diesen Kulturen präsentiert. Sie beeinflussen die Effizienz in der Obstzüchtung enorm. Ihre Entwicklung wäre jedoch ohne Mendels Erkenntnisse nicht möglich gewesen. Beim Apfel, der bedeutendsten einheimischen Obstart, gibt es eine Vielzahl von wirtschaftlich inte­ressanten mendelnden Merkmalen. Einige von ihnen, wie der Säulenwuchs, die rote Fruchtfleischfarbe, das Kältebedürfnis zum Brechen der Winterknospenruhe, die Samenlosigkeit der Früchte sowie gefüllte Blüten für den Anbau als Ziergehölz und die Resistenz gegenüber Schorf und Mehltau sind phänotypisch einfach zu erfassen. Eine Selektion auf der Basis des Phänotyps ist bei diesen Merkmalen meist problemlos möglich, wenngleich sie mithilfe molekularer Marker noch effektiver gestaltet werden kann. Andere Merkmale, wie die Resistenz gegenüber Insekten, verschiedenen Lagerkrankheiten oder der bakteriellen Feuerbrandkrankheit sind nicht so einfach anhand des Phänotyps zu bestimmen. Hier sind für eine erfolgreiche Züchtung molekulare Marker unabdingbar. Das gilt auch für die rote Färbung der Fruchtschale. Bei Süßkirschen ist die Situation sehr ähnlich. Zu den mendelnden Merkmalen mit ökonomischer Bedeutung gehören hier neben der Selbstfertilität auch die Mehltauresistenz und die ­Farbe der Fruchtschale.The influence of Mendel on modern fruit breeding is undeniable. The inheritance rules he established have enabled breeders to design breeding programmes in a targeted manner to make predictions about possible success and to select offspring with improved trait combinations using molecular genetic markers in a cost-effective and efficient way. Using the example of the apple and the sweet cherry as representatives of pome and stone fruit, we highlight selected Mendelian traits that are of particular importance for breeding. In addition, the current status of molecular markers and genetic maps and their enormous influence in efficient fruit breeding are presented. However, molecular marker progress would not have been possible without Mendel's insights. In apples, the most important native fruit species, there is a large number of economically interesting Mendelian traits some of which are easy to phenotype. For example, columnar growth, red flesh colour, cold requirement for breaking winter bud dormancy, seedlessness of the fruits and double flowers for cultivation as an ornamental tree and resistance to scab and powdery mildew. Other traits, such as resistance to insects, and the bacterial disease fire blight and various storage diseases are not as straightforward to phenotype. This also applies to red colouration of the fruit skin. Nevertheless, in both situations, molecular markers are indispensable for successful breeding. This also applies to sweet cherries. In addition to self-fertility, powdery mildew resistance and fruit skin colour are among the Mendelian traits with economic significance in sweet cherry breeding

Characterization of genomic DNA sequence of the candidate gene for FB_Mfu10 associated with fire blight resistance in Malus species

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

Deciphering the Mechanism of Tolerance to Apple Replant Disease Using a Genetic Mapping Approach in a Malling 9 × <i>M. × robusta</i> 5 Population Identifies SNP Markers Linked to Candidate Genes

Apple replant disease (ARD) is a worldwide economic risk in apple production. Although several studies have shown that the wild apple accession Malus × robusta 5 (Mr5) is ARD-tolerant, the genetics of this tolerance have not yet been elucidated. A genetic mapping approach with a biparental population derived from contrasting parents involving molecular markers provides a means for marker-assisted selection of genetically complex traits and for determining candidate genes. In this study, we crossed the ARD-tolerant wild apple accession Mr5 and the ARD-susceptible rootstock ‘M9’ and analyzed the resultant progeny for ARD tolerance. Hence, a high-density genetic map using a tunable genotyping-by-sequencing (tGBS) approach was established. A total of 4804 SNPs together with 77 SSR markers were included in the parental maps comprising 17 linkage groups. The phenotypic responses to ARD were evaluated for 106 offspring and classified by an ARD-susceptibility index (ASI). A Kruskal–Wallis test identified SNP markers and one SSR marker on linkage groups (LG) 6 and 2 that correlated with ARD tolerance. We found nine candidate genes linked with these markers, which may be associated with plant response to ARD. These candidate genes provide some insight into the defense mechanisms against ARD and should be studied in more detail