Innovative multiplex and its evaluation for effective genotyping of wild cherry

Abstract

Highlights• We present simple and cost-efficient method of SSR genotyping in cherry species.• Seven of fifteen previously published primer sequences have been altered in order to produce complementary amplicons in pseudo 15-plex.• The effectiveness and discriminatory power of established multiplex were verified by SSRs analysis of 48 wild cherry trees. AbstractTrees from the family Rosaceae play an important role in forest and agricultural ecosystems. Therefore, they are often an object of interest for both forest and horticultural tree breeders. Here, we present the utilization of an effective microsatellite (SSRs) genotyping method for wild cherry (Prunus avium L.) and verified the discriminatory power of the presented multiplex by genotyping 48 genetically distinctive individuals (plus-trees). Concerned loci were previously proven to be cross-compatible among various cultivars of cherry, hence, the method could have a broader utilization beyond to the field of forestry. Our technique is based on post-PCR processing of 15 polymorphic SSRs loci amplified in three multiplex reactions with fluorescently labeled primers (6-FAM, VIC, PET and NED). All PCR products could be pooled and analyzed simultaneously (pseudo 15-plex). In order to make this approach feasible, we redefined sequences of several primers. Thus, utilizing modified primers provides non-overlapping amplicons of each fluorescent dye.