Sweet cherry cultivar fingerprinting using single nucleotide polymorphisms detected by high resolution melt analysis

The Summerland breeding program has a long history of producing unique sweet cherry cultivars with a variety of desirable characteristics. Recent successes include the licensing of Sentennial, Sovereign and Staccato, which are progeny of Sweetheart, an earlier Summerland cultivar. Plant Breeders Rights for these cultivars are currently held by the Okanagan Plant Improvement Corporation (PICO) with detailed descriptors of the cultivars as the defining characteristics. We are currently developing molecular techniques to discriminate between these very closely related cultivars. Previously, AFLP and SSR analysis have successfully differentiated most cherry cultivars. However, only one SSR and one AFLP marker distinguished Sweetheart from Staccato. The requirement for more unique markers led to development of our current SNP fingerprinting strategy. New single nucleotide polymorphisms (SNPs) were identified from analysis of Illumina RNA-Seq libraries generated for Sweetheart and its three progeny. High coverage Sweetheart SNPs were examined for segregation in the progeny and confirmed by High Resolution Melt (HRM) assays. A set of 16 SNPs was chosen for independent segregation across the genome that provided a unique fingerprint of Sweetheart and each of its progeny. The straightforward SNP HRM technique was further tested on a broader set of 35 cultivars of interest to PICO, providing unique fingerprints for each. Within this group of 35 cultivars, 16 were selected for genotyping-by-sequencing to identify additional SNP markers. The choice of multiple unlinked SNPs increases the probability that each pattern of inheritance will be unique and can be used to verify the identity of unknown cultivars.