Characterization of genetic diversity in germplasm collections is fundamental for an efficient management and utilization. The citrus germplasm in the Western Caucasus represent one of the most northerly citrus collections. The collection contains numerous cultivars specifically selected for frost-tolerance, which are valuable as genetic resources for citrus breeding programs worldwide. Currently, the field citrus collection consists of more than 200 accessions, but their taxonomic assignment, genetic structure and diversity has not been clarified. In our study, 130 different citrus accessions including 24 species or interspecific hybrids maintained and bred at the Federal Research Centre the Subtropical Scientific Centre of the Russian Academy of Sciences (FRC SSC RAS) were genetically investigated using 13 biparental (ncSSR) and 13 uniparental (orgDNA) markers. After STRUCTURE analysis, the single accessions were allocated to eight genetic clusters and their relationships were elucidated. This phylogenetic relationship was also confirmed by principal coordinates analysis (PCoA) and by dissimilarity analysis using DARwin. We identified twenty accessions as misclassified and reassigned them to the correct species. For the remaining 110 accessions the taxonomical assignment could be confirmed. A high genetic diversity was observed on basis of the nuclear ncSSR markers, while the detection of 19 different haplotypes allowed the distinct differentiation of the accessions within our collection. Our study showed that the combination of ncSSR and orgDNA was an efficient tool to estimate the genetic diversity, genetic structure and phylogeny in the FRC SSC RAS citrus germplasm collection. The results obtained are valuable for future management of the collection and the usage of individual accessions as genetic resource for citrus cultivar and rootstock breeding, especially in colder regions.

Abstract This chapter discusses the botany and history, importance, breeding and genetics, molecular genetics, genomics, functional genomics, gene cloning, gene mapping, micropropagation, somatic cell genetics, genetic manipulation, somatic hybridization, genetic transformation, and cryopreservation of apple.

Raspberry (Rubus idaeus L.) cultivars are propagated vegetatively, as many other fruit species from the Rosaceae family. During propagation, mistaken identities of cultivars can be caused by mislabelling plants for various reasons. This poses a problem not only for growers who buy cultivars because of specific characteristics (e.g., resistance) but also for breeders who are being deprived of licensing fees for cultivars under Plant Variety Protection (PVP). In this study, six raspberry cultivars of up to six different origins were tested for trueness-to-type by fingerprinting with 16 SSR markers. Nine out of 33 samples turned out not to be true-to-type, seven from online shops and two from nurseries.

Apple replant disease (ARD), also described as soil decline, is known for centuries; however, up to now the ultimate causes are unknown. After replanting plants of the same species, the plants are characterized by a poor vegetative development, stunted growth and reduced yield in terms of quantity and quality. For tree nurseries and orchards, replant disease is increasingly problematic and a sustainable and economically feasible solution would be the use of ARD-tolerant rootstocks. Up to now, only a few Malus genotypes with tolerance to ARD have been described and hardly any information is available about the genetic and physiological mechanisms underlying these tolerances. The discrimination of ARD tolerant and susceptible genotypes is a prerequisite for future rootstock breeding, but also for molecular studies aiming at improving the breeding efficiency. For this reason, 48 genotypes of wild apple species and rootstock cultivars were evaluated for their tolerance/susceptibility to ARD in a greenhouse bio-test. The susceptibility to ARD was classified by an ARD susceptibility index (ASI) calculated based on biomass and the increase in shoot length, respectively. The development of genetic linkage maps enables the detection of markers linked to agronomical important traits. Such associated markers can be used for an early and rapid selection of eligible plants during the breeding process. In order to map quantitative trait loci (QTL) for ARD tolerance a cross between the genotype Malus x robusta 5 (tolerant to ARD) and the rootstock M.9 (susceptible to ARD) was established and used for the construction of a first genetic linkage map including 78 SSR anchor markers. In addition, a comparative transcriptome study using mRNA from roots of M.9 and M.xrobusta 5, which were cultivated on both ARD and disinfected ARD soil will provide information about potential candidate genes, which are related to ARD-tolerance.

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The 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 cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.

Biennial or alternate bearing in fruit trees leads to fluctuating yields, characterized by ‘On’ years with high crop loads followed by ‘Off’ years with little or no yield. To study the mechanism of biennial bearing in apple (Malus domestica Borkh.), the cultivars ‘Royal Gala’ as a regular bearer and ‘Fuji’ as a strongly alternating cultivar were investigated. ‘Off’ trees were completely thinned at full bloom, whereas ‘On’ trees were not thinned to maintain the natural high crop load. The first signs of floral bud initiation were detected for ‘Fuji’ at 70 days after full bloom (DAFB) and 120 DAFB for ‘Off’ and ‘On’ trees, respectively. In ‘Royal Gala’, initiated buds were observed not before 100 DAFB in both treatments. Hence, buds from ‘Fuji’ ‘Off’ trees committed to flowering about one month earlier than those from ‘Royal Gala’ ‘Off’ trees. In contrast, flower initiation of buds from ‘Royal Gala’ ‘On’ trees occurred 20 days earlier when compared to those from ‘Fuji’ ‘On’ trees. Electrospray ionization mass spectrometry (ESI-MS) coupled with ultra-high performance liquid chromatography (UHPLC) allowed the identification of 3011 proteins. A Student’s T-Test revealed 181 differentially expressed proteins between buds from ‘On’ and ‘Off’ trees. Specifically, 81 proteins were significantly upregulated in floral buds and 100 proteins in vegetative buds, respectively. The largest log2 fold change of 3.17 was identified for a zinc metalloprotease in buds from the ‘Fuji’ ‘On’ treatment. The protein with the highest up-regulation in ‘Fuji’ ‘Off’ buds was a nigrin b-like protein. Histological analysis of bud development has proven to be an essential and reliable tool to identify the time point of floral initiation in apple. Moreover, this study demonstrates that especially in biennial bearing cultivars, crop load heavily influences the onset of floral bud initiation and can differ for up to 50 days within the same cultivar depending on crop load. The identification of this critical period enables in-depth analysis of biochemical changes such as differences in protein profiles to further reveal the mechanism of biennial bearing in apple.

The apple wild species accession Malus fusca MAL0045 had been found to be resistant to fire blight in artificial inoculation trials with Erwinia amylovora strain Ea222_JKI. Consequently, using a population derived by crossing MAL0045 with Idared, the corresponding fire blight resistance locus of M. fusca (Mfu10) was mapped on chromosome 10 explaining up to 66% of phenotypic variation at a logarithm of the odd (LOD) ratio of 31.0 with the strain Ea222_JKI. Moreover, the very aggressive strain Ea3049 only minimally affected MAL0045 but significantly affected the population although could not overcome the resistance of Mfu10. To further understand the resistance mechanism of M. fusca, we evaluated resistance of the original mapping population, comprised of 134 individuals, to E. amylovora strain ZYRKD3-1 which causes the breakdown of the resistance of M.xrobusta 5 (Mr5) and the Mr5 fire blight resistance QTL on LG3. Our results showed that the major QTL of M. fusca on LG10 could still be detected at the same exact position with a higher effect on fire blight resistance, indicating that ZYRKD3-1 has no effect on Mfu10, although the mean percent lesion length of the population was almost doubled compared with Ea222_JKI.

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Gene-bank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis 'Yunnan', F. vesca 'Illa Martin' and F. moschata 'Bauwens' were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9. C and the GFP-tagged strain XF3.9. C-(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post-inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.

The grey mould disease caused by Botrytis cinerea leads to substantial economic losses in strawberry production all over the world. Control of the disease requires an extensive amount of fungicide that is applied in varying complexes because the pathogen easily develops resistance against the active compounds. Planting of resistant cultivars seems to be a promising alternative for fruit growers, but there are currently no cultivars available combining resistance to B. cinerea with attractive horticultural traits. Breeding of new cultivars requires the effective identification of resistant strawberry genotypes; therefore the current study was aimed at the evaluation of strawberry genetic resources under controlled conditions by establishing an artificial inoculation assay. The method presented in this study is an artificial inoculation of ripe fruits with a defined spore suspension under laboratory conditions. The results show that this assay is fast and simple and leads to reproducible results that correlate with field observations. Over 3 years a total of 107 strawberry genotypes of the German National Fruit Genebank at the JKI in Dresden‐Pillnitz were evaluated. Five partly resistant genotypes, cultivars Diana, Joerica and Kimberly, and Fragaria virginiana ‘Wildmare Creek’ and F. vesca subsp. bracteata, were identified with mean disease levels of <20% at 6 days post‐inoculation. The obtained results are discussed with regard to future breeding activities.

The preservation of fruit genetic resources is a pre-requisite for ensuring a sustainable fruit production for future generations. In Germany, the preservation of fruit genetic resources has a long-lasting tradition, which reaches back to the Middle Ages. In the early decades of the 20th century national collections were established as a basis for scientifically sound breeding programmes. Since that time, a multitude of genotypes of different fruit crop species are preserved. Preservation is done in the aforementioned public germplasm collections and in private collections as well. Unfortunately, there is currently nobody, who has a general overview of all the ongoing activities. The loss of individual genotypes, which are only preserved in one or a few collections, can therefore not be excluded. On this account the German National Fruit Genebank has been established in 2009 to minimize the risk of losing fruit genetic resources. The German National Fruit Genebank is a decentral network, which is aimed on the coordination of different germplasm collections. The work is organized in species specific networks, where preservation is collectively performed. Networks for strawberry, cherry, apple and plum are already established. Two further networks for Rubus-species and pear are currently established. The establishment of networks for further fruit crops has been scheduled. All genotypes will be investigated for trueness-to-type using morphological characters and DNA fingerprints.

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.

Fire blight, which is caused by the bacterial pathogen Erwinia amylovora, is one of the most devastating diseases in apple production. Breeding for fire blight resistance is therefore one of the major aims in nearly all apple breeding programs. In this study a QTL mapping approach for the cross population 'Idared' x Malus xrobusta 5 (Mr5) was performed after inoculation with E. amylovora strains differing in their virulence to Mr5 to detect major and minor QTLs. The major QTL on LG3 of Mr5 was stable for all inoculations with strains inducing resistance of Mr5 whereas inoculation with all strains breaking resistance of Mr5 resulted in a breakdown of the QTL on LG3. The results gave further evidence for the existence of a gene-for-gene relationship and demonstrated clearly that the fire blight resistance of Mr5 is strain specific. Several minor QTLs were detected on different linkage groups of Mr5. Additionally, there are indications that Malus baccata 'Jackii' acts in a similar manner like Mr5 regarding resistance to fire blight.

Fire blight, caused by Erwinia amylovora, is a major concern in apple production since no existing control option has been proven to be completely effective and durable. The use of resistant apple cultivars in apple production could and should be an importance piece of an integrated management of the disease. High levels of resistance to Erwinia amylovora have been found in wild or ornamental apples like 'Evereste'. Recent studies showed that this resistance is largely controlled by a single QTL. However, no advanced selections carrying this QTL (Fb_E locus) and displaying satisfactory fruit quality and agronomic performance are yet available. In order to remove all the undesired traits present in a wild apple up to five (pseudo) backcrosses are necessary. Considering that the juvenile phase of apple lasts at least 4-5 years, this can take 20-25 years. Recently, a breeding technology based on the early flowering BpMADS4-transgenic line T1190 was presented. Using this line, the selection of pre-breeding genotypes carrying the Fb_E locus was started in 2009. In each generation foreground selection using molecular markers associated with the locus Fb_E and the transgene BpMADS4 was used to identify the seedlings to consider as parents in the next crosses once these started producing flowers (generally within 2 to 5 months after seed planting). Starting from the BC'(1) generation, background selection based on SSR markers was also applied on early flowering Fb_E seedlings. The current status of the introduction of the Fb_E locus as well as observations and methods applied to increase the efficiency of the approach will be presented.

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 xrobusta 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.

After fire blight inoculation, shoots of apple and pear formed biphenyls and dibenzofurans as defense compounds. The phytoalexins were only present in the transition zone of stems, whereas leaves were devoid of these compounds. In shoots of the apple cultivar 'Holsteiner Cox', four biphenyls and two dibenzofurans accumulated in the downward advancing transition zone six weeks after Erwinia amylovora inoculation. In the pear cultivar 'Conference', three biphenyls and one dibenzofuran were detected. The total phytoalexin content in the transition zone of pear was much lower than that in apple. In addition to 'Conference', two other pear cultivars, 'Alexander Lucas' and 'Harrow Sweet' were investigated for their phytoalexin formation in response to fire blight inoculation. Differences in the morphological changes as well as in the biphenyl and dibenzofuran concentration and pattern were observed between these three genotypes. 'Harrow Sweet', classified as most resistant to fire blight, showed the highest phytoalexin concentration when compared to 'Alexander Lucas' and 'Conference' and its transition zone stopped the downward migration 12 days after inoculation. Five biphenyls and one dibenzofuran were detectable in this pear cultivar.

In Germany, all activities of fruit genetic resources management are based on the German National Program for Genetic Resources of Agricultural and Horticultural Plants. Three areas of actions, like ex situ conservation in field collections and building safety back-ups, characterization of the material, and utilization of genetic resources in breeding are described for fruit species collected at Dresden-Pillnitz.

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.

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.

Swiss and more generally European apple (Malus × domestica) production is hampered by several diseases, the most destructive being fire blight, caused by Erwinia amylovora. On the other hand, there are apple scab, caused by Venturia inaequalis and powdery mildew, caused by Podosphaera leucotricha, which represent the major phytosanitary problems. Classical breeding has produced many scab and mildew resistant cultivars and efforts to breed also fire blight resistant cultivars are currently undertaken. Marker assisted selection (MAS) increases efficiency by allowing early non-destructive screening of seedlings and identifying genotypes showing pyramids of resistance genes. If the development of markers for MAS was the primary goal of genetic analysis in the 1990s, identification and cloning of resistance genes is now the goal. The first and until now the sole resistance gene which has been isolated and transformed into a susceptible apple cultivar is the gene HcrVf2 (Rvi6), responsible for the Vf scab resistance present in most classically bred scab resistant cultivars. Much effort is currently spent in the identification and positional cloning of other apple genes conferring resistance to apple scab and fire blight. In our labs, we identified the putative scab resistance gene Rvi15 and two fire blight resistance genes namely from ‘Evereste’ and Malus × robusta 5. The functionality of these candidate genes is currently under scrutiny by complementation experiments. However, the final goal is the creation of a product, e.g., an improved apple cultivar that is resistant to scab and fire blight. The ideal product would have advantages to the environment and producer, and should raise as little concern as possible with consumers. To accomplish this ‘ideal product’, we opted for the cisgenic approach by introducing the scab resistance gene HcrVf2 with its own regulatory sequences into the highly susceptible apple cultivar, ‘Gala’, through Agrobacterium transformation. All marker genes were eliminated after transformation. Similarly, we are currently introducing into both the readily developed cisgenic ‘Gala’ and in the untransformed ‘Gala’ the putative Malus own fire blight resistance gene candidates, aiming at both proof of functionality of the identified candidates and possibly at rapid development of a fire blight and scab resistant cisgenic apple.

Organic strawberry production suffers from high yield losses caused by numerous fungal and bacterial diseases. Two of the most important diseases are the grey mould disease caused by Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana), and the bacterial angular leaf spot disease caused by Xanthomonas fragariae (Kennedy & King). Beside cultivation methods and organic plant protection measures, the development of resistant cultivars seems to be the most promising strategy in order to improve the productivity in organic strawberry cultivation. Therefore, we established resistance tests to determine resistant and susceptible strawberry cultivars and breeding selections. In a first run, 40 different cultivars and selections were tested for their susceptibility towards B. cinerea by artificial inoculation of fruits and leaves and evaluation of the disease symptoms. Plants of 40 cultivars were tested for susceptibility to X. fragariae by artificial inoculation in the greenhouse. In a diallel crossing approach, 12 commonly cultivated strawberry cultivars have been crossed reciprocally and propagated in a field trial. Important characteristics of the progeny such as ripening time, yield, morphological traits and occurrence of diseases have been evaluated for a period of two consecutive years and lead to the determination of general (GCA) and specific (SCA) combining abilities. Together with the results of the resistance tests we identified a set of genotypes that show resistant characteristics towards B. cinerea and might be suitable for use in organic cultivation systems. Furthermore, they can be used for targeted breeding experiments in the future.