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Apple cultivars and breeding lines that represent much of the diversity currently present in major European breeding programmes and are genetically related by their pedigree were examined for the trueness of their identity and parentage by consistency in marker scores using a genome-covering set of 80 microsatellite (SSR) markers and an 'identity-by-descent' approach. One hundred and twenty-five individuals were validated for the trueness-to-type of both their parents and 49 were validated for one of their parents, their second being unknown (23 individuals) or not available in this study (26 individuals). In addition, 15 individuals for which we lacked one of or both the direct parents were validated by consistency with tested parents of earlier generations. Furthermore, the identity of 28 founder cultivars was validated, their marker scores being consistent with descending cultivars and breeding lines. Four of the eight triploids identified were clearly shown to have arisen from unreduced egg cells. The assumed pedigree of 15 further individuals was found to be incorrect; fully consistent pedigrees were suggested for three of the cultivars. The pedigrees of a further eight individuals were confirmed through inference from the molecular data. [ABSTRACT FROM AUTHOR]

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented. [ABSTRACT FROM AUTHOR]

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping projects. [ABSTRACT FROM AUTHOR]

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database () to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM. [ABSTRACT FROM AUTHOR]

Breeding for scab-resistant apple cultivars by pyramiding several resistance genes in the same genetic background is a promising way to control apple scab caused by the fungusVenturia inaequalis. To achieve this goal, DNA markers linked to the genes of interest are required in order to select seedlings with the desired resistance allele combinations. For several apple scab resistance genes, molecular markers are already available; but until now, none existed for the apple scab resistance geneVbjoriginating from the crab appleMalus baccata jackii. Using bulk segregant analysis, three RAPD markers linked toVbjwere first identified. These markers were transformed into more reliable sequence-characterised amplified region (SCAR) markers that proved to be co-dominant. In addition, three SSR markers and one SCAR were identified by comparing homologous linkage groups of existing genetic maps. Discarding plants showing genotype-phenotype incongruence (GPI plants) plants, a linkage map was calculated.Vbjmapped between the markers CH05e03 (SSR) and T6-SCAR, at 0.6 cM from CH05e03 and at 3.9 cM from T6-SCAR. Without the removal of the GPI plants,Vbjwas placed 15 cM away from the closest markers. Problems and pitfalls due to GPI plants and the consequences for mapping the resistance gene accurately are discussed. Finally, the usefulness of co-dominant markers for pedigree analysis is also demonstrated. [ABSTRACT FROM AUTHOR]

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers. [ABSTRACT FROM AUTHOR]

The availability of suitable genetic markers is essential to efficiently select and breed apple varieties of high quality and with multiple disease resistances. Microsatellites (simple sequence repeats, SSR) are very useful in this respect since they are codominant, highly polymorphic, abundant and reliably reproducible. Over 140 new SSR markers have been developed in apple and tested on a panel of 7 cultivars and 1 breeding selection. Their high level of polymorphism is expressed with an average of 6.1 alleles per locus and an average heterozygosity (H) of 0.74. Of all SSR markers, 115 have been positioned on a genetic linkage map of the cross ‘Fiesta’ × ‘Discovery’. As a result, all 17 linkage groups, corresponding to the 17 chromosomes of apple, were identified. Each chromosome carries at least two SSR markers, allowing the alignment of any apple molecular marker map both with regard to identification as well as to orientation of the linkage groups. To test the degree of conservation of the SSR flanking regions and the transferability of the SSR markers to other Rosaceae species, 15 primer pairs were tested on a series of Maloideae and Amygdaloideae species. The usefulness of the newly developed microsatellites in genetic mapping is demonstrated by means of the genetic linkage map. The possibility of constructing a global apple linkage map and the impact of such a number of microsatellite markers on gene and QTL mapping is discussed. [ABSTRACT FROM AUTHOR]

Post-meiotic mutants affecting pollen development are fundamental tools for defining the genetic program controlling microsporogenesis and pollen function. An example of such mutants is gametophytic male sterile-1 ( gaMS-1). Heterozygous plants gaMS-1/+ that have a normal phenotype and are female fertile, segregate 1:1 normal:sterile pollen grains and their selfed progeny segregates 1:1 normal:semi-sterile plants. With the final aim of isolating the gene, a positional cloning strategy was adopted. In this paper, we report the results of fine mapping GaMS-1 by different types of molecular markers. Two back crosses were used as mapping populations. They were obtained by crossing the line carrying the mutation with the inbred lines Mo17 and WF9, used as recurrent male parents. Linkage disequilibrium analysis allowed assigning GaMS-1 to the short arm of chromosome 2.By the combined use of SSR, AFLP, PCR markers and ESTs a region of 1 cM containing GaMS-1 was delimited. [ABSTRACT FROM AUTHOR]

A positional cloning project was started in apple with the aim of isolating the Vf resistance gene of Malus floribunda 821. Vf confers resistance against apple scab, the most important disease in apple orchards. A chromosome walk starting from two molecular markers (M18-CAPS and AM19-SCAR) flanking Vf was performed, using a bacterial artificial chromosome (BAC) library containing inserts of the cultivar Florina, which is heterozygous for Vf. Thirteen BAC clones spanning the region between the two markers were identified in nine chromosome walking steps. The size of the resulting contig is approximately 550 kb. In order to map the Vf region in more detail, we analyzed over 2000 plants from different populations segregating for Vf with markers produced from BAC end sequences. In this way, we were able to restrict the possible location of the Vf gene to a minimum of five clones spanning an interval of approximately 350 kb. [ABSTRACT FROM AUTHOR]

A map-based cloning scheme is being used to isolate the Vf resistance gene of apple against the fungus Venturia inaequalis. Vf is a major dominant gene that is inherited in a Mendelian manner and influenced by minor genes that modify its activity. The two recently published markers M18 and AL07, bracketing Vf, were tested on 1179 progeny plants of three crosses to fine-map Vf. M18 and AL07 were positioned at 0.2 cM and 1.1 cM from Vf respectively, for a total distance between the two markers of 1.3 cM. Physical mapping by pulsed-field gel electrophoresis, using M18 and AL07 as probes, demonstrated that both markers hybridize to a common 870 kb NotI restriction fragment. We therefore found a relationship between physical and genetic distance of 670 kb/cM in the Vf region. This led us to the conclusion that a chromosome walk using the recently published apple BAC library starting from M18 and AL07 is feasible. [ABSTRACT FROM AUTHOR]

Breeding resistant apple plants is an alternative way to control fungal pathogens reducing the environmental impact due to the use of pesticides. The breeding of apple cultivars resistant to Venturia inaequalis could be much improved by marker-assisted selection. A molecular marker closely linked to the resistance locus called Vf could replace selection based on infection studies. To find such molecular markers, DNA of progenies from crossings of a resistant and a susceptible apple tree was subject to bulked segregant analysis. Two markers were found with a genetic distance of 10.6% and 19.7% recombination frequency to the Vf locus. [ABSTRACT FROM AUTHOR]

The development of highly informative markers, such as simple sequence repeats, for tagging genes controlling agronomic characters is essential for apple breeding. Furthermore the use of these markers is fundamental both for variety identification and for the characterisation and management of genetic resources. We have developed 16 reliable simple sequence repeat (SSR) markers that amplify all alleles from a panel of 19 Malus x domestica (Borkh.) cultivars or breeding selections and from Malus floribunda 821. Those markers show a high level of genetic polymorphism, with on average 8.2 alleles per locus and an average heterozygosity of 0.78. Due to this high level of polymorphism, it was possible using two selected SSRs to distinguish all cultivars except Starking and Red Delicious. Ten of the markers we developed have been mapped on a RAPD linkage map, proving their Mendelian segregation as well as their random distribution in the apple genome. Finally, we discuss the importance of using co-dominant markers in outbreeding species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Densely saturated genetic maps of neutral genetic markers are a prerequisite either for plant breeding programs to improve quantitative traits in crops or for evolutionary studies. cDNA and genomic clones from maize were utilized to initiate the construction of a RFLP linkage map in Sorghum bicolor. To this purpose, an F population was produced from starting parental lines IS 18729 (USA) and IS 24756 (Nigeria) that were differentiated with regard to many morphological and agronomical traits. A total of 159 maize clones were hybridized to the genomic DNA of the two parents in order to detect polymorphism: 154 probes hybridized to sorghum and 58 out of these were polymorphic. In almost all of the cases hybridization patterns were similar between maize and sorghum. The analysis of the segregation of 35 polymorphic clones in an F population of 149 individuals yielded five linkage groups. The three principal ones recall regions of maize chromosomes 1, 3 and 5: in general, colinearity was maintained. A possible inversion, involving a long region of maize chromosome 3, was detected. Simulations were also performed to empirically obtain a value for the lowest number of individuals of the F population needed to obtain the same linkage data. [ABSTRACT FROM AUTHOR]