The Development of New SSR Markers and an Assay for Genotyping Sweet Cherry (Prunus avium L.) in One Reaction

Sweet cherry (Prunus avium L.) exhibits relatively low genetic diversity because of the self-compatibility of some varieties and repeated crossings of the same genotypes. High-quality markers are therefore needed for their reliable discrimination. However, the most currently used simple sequence repeat (SSR) markers offer only limited resolution for genotyping purposes. Here, thirty new highly polymorphic SSR markers were extracted from whole-genome sequences of 299 sweet cherry genotypes. Then, 16 highly polymorphic SSR markers were selected, multiplexed into one PCR, and successfully verified on a collection containing 294 unique genotypes. Compared with the set of 16 SSR markers recommended by the European Cooperative Programme for Plant Genetic Resources (ECPGR) for sweet cherry genotyping, our newly developed system has a seven orders of magnitude lower probability of the random identity of two genetically distinct samples than the ECPGR set (10^-19 vs. 10^-12). This higher resolution not only enables more precise genotyping but can also be successfully used for parentage or population analyses. This new and unique one-tube approach for sweet cherry genotyping will substantially simplify genotyping workflows, minimize errors, and save labor, time, and cost.