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