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 project

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 dat

Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids

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 project

Development of a set of apple ssrs markers spanning the apple genome, genotyping of HiDRAS Plant material and validation of genotypic data

The Pedigree Genotyping concept (van de Weg et al., 2004) forms the base of the EU-project HiDRAS, aimed at the identification of molecular markers for fruit quality and disease resistance. The concept allows the exploitation of breeding material in the assessment of marker-trait associations and in allele mining by using multiple pedigreed plant populations, which can be any combination of crosses, cultivars, and breeding selections. The Pedigree Genotyping model makes use of directed genotyping and the so-called Identity By Descent (IBD) concept. Prerequisites for this innovative technique are a good coverage of the apple genome with highly polymorphic simple sequence repeats (SSR) markers, and consistent molecular data throughout the whole pedigree. At the beginning of the HiDRAS project, about 160 apple SSRs were available and mapped. However the coverage of the apple genome was not sufficient as there were several regions of the genome with no SSRs available, or the level of polymorphism was low. To fill these gaps with new SSRs, two approaches were used: i) development and mapping of SSRs from SSR-enriched genomic libraries and available apple EST sequences containing repeated motifs; and ii) making use of the synteny existing between apple and pear, by using SSR markers developed in pear. These strategies enriched the apple reference map, 'Fiesta' x 'Discovery', with 156 new SSRs. A subset of 88 reliable SSR markers, highly polymorphic and evenly distributed across the apple genome, was selected. Eighty-three out of 88 SSR markers were organized in 21 multiplex PCR reactions and used to genotype the HiDRAS plant material (around 2000 DNA samples representing over 1750 genotypes). After the generation of raw data, each single data point was verified for consistency throughout the whole pedigree by the use of newly developed software packages. This allowed the identification of not only scoring errors, but also mistakes in the reported pedigrees and the "(not) true-to-typeness" of some genotypes (wrong plants). The complete procedure from SSR development to data validation is presented

Development of a set of apple SSRs markers spanning the apple genome, genotyping of HiDRAS plant material and validation of genotyping data

International audienceThe Pedigree Genotyping concept (van de Weg et al., 2004) forms the base of the EU-project HiDRAS, aimed at the identification of molecular markers for fruit quality and disease resistance. The concept allows the exploitation of breeding material in the assessment of marker-trait associations and in allele mining by using multiple pedigreed plant populations, which can be any combination of crosses, cultivars, and breeding selections. The Pedigree Genotyping model makes use of directed genotyping and the so-called Identity By Descent (IBD) concept. Prerequisites for this innovative technique are a good coverage of the apple genome with highly polymorphic simple sequence repeats (SSR) markers, and consistent molecular data throughout the whole pedigree. At the beginning of the HiDRAS project, about 160 apple SSRs were available and mapped. However the coverage of the apple genome was not sufficient as there were several regions of the genome with no SSRs available, or the level of polymorphism was low. To fill these gaps with new SSRs, two approaches were used: (i) development and mapping of SSRs from SSR-enriched genomic libraries and available apple EST sequences containing repeated motifs; and (ii) making use of the synteny existing between apple and pear, by using SSR markers developed in pear. These strategies enriched the apple reference map, 'Fiesta' * 'Discovery', with 156 new SSRs. A subset of 88 reliable SSR markers, highly polymorphic and evenly distributed across the apple genome, was selected. Eighty-three out of 88 SSR markers were organized in 21 multiplex PCR reactions and used to genotype the HiDRAS plant material (around 2000 DNA samples representing over 1750 genotypes). After the generation of raw data, each single data point was verified for consistency throughout the whole pedigree by the use of newly developed software packages. This allowed the identification of not only scoring errors, but also mistakes in the reported pedigrees and the "(not) true-to-typeness" of some genotypes (wrong plants). The complete procedure from SSR development to data validation is presented

Bayesian QTL analyses using pedigreed families of an outcrossing species, with application to fruit firmness in apple

Bayesian QTL linkage mapping approaches offer the flexibility to study multiple full sib families with known pedigrees simultaneously. Such a joint analysis increases the probability of detecting these quantitative trait loci (QTL) and provide insight of the magnitude of QTL across different genetic backgrounds. Here, we present an improved Bayesian multi-QTL pedigree-based approach on an outcrossing species using progenies with different (complex) genetic relationships. Different modeling assumptions were studied in the QTL analyses, i.e., the a priori expected number of QTL varied and polygenic effects were considered. The inferences include number of QTL, additive QTL effect sizes and supporting credible intervals, posterior probabilities of QTL genotypes for all individuals in the dataset, and QTL-based as well as genome-wide breeding values. All these features have been implemented in the FlexQTL\u2122 software. We analyzed fruit firmness in a large apple dataset that comprised 1,347 individuals forming 27 full sib families and their known ancestral pedigrees, with genotypes for 87 SSR markers on 17 chromosomes. We report strong or positive evidence for 14 QTL for fruit firmness on eight chromosomes, validating our approach as several of these QTL were reported previously, though dispersed over a series of studies based on single mapping populations. Interpretation of linked QTL was possible via individuals\u2019 QTL genotypes. The correlation between the genomic breeding values and phenotypes was on average 90 %, but varied with the number of detected QTL in a family. The detailed posterior knowledge on QTL of potential parents is critical for the efficiency of marker-assisted breeding