W.E. van de Weg

Development and characterisation of 140 new microsatellites in apple (Malus x domestica Borkh.)

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.

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F1 progeny derived from a cross between the apple cultivars ‘Discovery’ and ‘TN10-8’ were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica).

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I–III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions.

The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple.

Russian apple R12740-7A is the designation for an accession grown from seed collected in Russia, which was found to be highly resistant to apple scab. The resistance has historically been attributed to a naturally pyramided complex involving three major genes: one race-nonspecific gene, Vr, conditioning resistance to all known races, plus two race-specific genes. The race-nonspecific gene was identified as an independently segregating gene by Dayton and Williams (1968) and is referred to in this paper as Vr-DW. The first researchers to study the scab resistance gene complex in Russian apple never described the phenotype conditioned by the race-nonspecific gene. Later, Aldwinckle et al. (1976) associated the name Vr with a scab resistance gene conditioning distinctive stellate necrotic reactions, which we refer to as Vr-A in order to distinguish it from Vr-DW. We show that the segregation ratios in progenies from the scab differential hosts 2 and 4 that are derived from Russian apple, crossed with susceptible cultivars were consistent with a single gene conditioning resistance in each host. The genes have been named Vh2 and Vh4, respectively. Resistant segregants from host 2 showed stellate necrotic reactions, while those from host 4 showed hypersensitive reactions. Both the phenotypes and the genetic maps for the genes in the respective hosts were very similar to those of the genes previously named Vr-A and Vx, respectively, in an F1 family of Russian apple. We showed that race 2 of V. inaequalis isolated from host 2 was able to infect resistant descendants of the non-differential accession PRI 442-23 as well as host 2. The descendants of PRI 442-23 were expected to carry the race-nonspecific Vr-DW gene, but in fact carry Vr-A. We conclude that the Vh2 gene in host 2 and Vr-A are the same, and that the Vh4 gene in host 4 and Vx are the same. However, a major finding of this study is that the latter gene mapped to linkage group 2 of apple instead of linkage group 10 as suggested from previous research. With the two race-specific genes from Russian apple defined now, we discuss the nature of the race-nonspecific Vr-DW gene in this accession. We also report the identification of a new scab resistance gene, VT57, from either ‘Golden Delicious’ or ‘Red Dougherty’, which conditions chlorotic resistance reactions and is linked to Vh2.

Bayesian analysis of complex traits in pedigreed plant populations

A Bayesian approach to analyze complex traits is presented that can help plant eneticists and breeders in exploiting the marker and phenotypic data on pedigreed populations as available from ongoing breeding programs. The statistical model for the quantitative trait may include non-genetic and genetic components. The latter component can be divided into QTL on known marker linkage groups, major genes and a polygenic component. The full probability model, prior assumptions on model variables are presented and criterion for model selection and posterior inferences are given. Simulated data on a known pedigreed population structure of the EU project HiDRAS was used to illustrate the use of the Bayesian approach to analyze complex traits. It was shown that estimates for QTL parameters were more accurate when non-genetic factors were included in the model and when a polygenic component was included when not all linkage groups were analyzed simultaneously. The Bayesian approach has been implemented into the software package FlexQTL and allows plant breeders explore their pedigreed populations for segregating QTL alleles that are relevant in their breeding program.

Map position and functional allelic diversity of Md-Exp7, a new putative expansin gene associated with fruit softening in apple (Malus × domestica Borkh.) and pear (Pyrus communis)

Fruit ripening can be considered as a complex set of biochemical and physiological changes occurring at the end of the developmental stage. Ripe fruit texture notably affects overall quality and consumer appreciation. Excessive softening limits shelf-life and storability, thereby increasing disease susceptibility and economic loss. Fruit softening is a process due to the depolymerisation of different polysaccharide classes, an event controlled by a synergic and coordinated action of several enzymes among which expansins play a fundamental role. To date, six expansin genes are known to be expressed during apple fruit ontogeny, from full bloom up to fruit ripening. We identified a novel expansin apple homolog (Md-Exp7) sharing high sequence similarity with specific-ripening expansin genes of other crops. A functional marker (Md-Exp7SSR) based on an SSR motif located within the untranslated region of the gene was developed and mapped on Linkage Group 1 of the apple and pear genomes in a region where one major apple QTL for fruit firmness had been previously identified. The allelic composition of 31 apple varieties for the SSR marker was associated with differences in fruit softening.

Genotyping of pedigreed apple breeding material with a genome-covering set of SSRs: trueness-to-type of cultivars and their parentages

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.

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

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.

Genomic characterization and linkage mapping of the apple allergen genes Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin)

Four classes of apple allergens (Mal d 1, −2, −3 and −4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene (Mal d 2.01A and Mal d 2.01B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01A and Mal d 2.01B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes (Mal d 4.01A and B, Mal d 4.02A and Mal d 4.03A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02A and Mal d 4.03A were single copy genes located on linkage group 2 and 8, respectively.

Identification of RAPD markers linked to a Phytophthora fragariae resistance gene (Rpf1) in the cultivated strawberry

Abstract Bulked segregant analysis (BSA) was used to identify seven random amplified polymorphic DNA (RAPD) markers linked to the Rpf 1 gene. Rpf 1 confers resistance to Phytophthora fragariae var. fragariae, the causal agent of red stele root rot in Fragaria spp. The bulked DNAs represented subsets of a F1 population obtained from the cross Md683×Senga Sengana which consisted of 60 plants and segregated in a 1:1 ratio for resistance or susceptibility to race 2.3.4 isolate NS2 of P.  fragariae. Seven markers were shown to be linked to Rpf 1 and were generated from four primers; five of these markers were in coupling phase and two in repulsion phase with respect to the gene. A linkage map of this resistance gene region was generated using JoinMap 2.0TM. The manner in which Rpf 1 and the linked markers co-segregated indicated that they are inherited in a disomic fashion. These markers could enable gene pyramiding and marker-assisted selection of resistance genes in strawberry breeding programmes.