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.