Development of High Quality EST-SSR Markers Without Stutter Bands in Peach and Their Application in Cultivar Discrimination and Hybrid Authentication

Peach (Prunus persica) is an important fruit crop worldwide with several thousand cultivars. Cultivar discrimination and hybrid authentication are often required in peach breeding and can be achieved by applying various molecular markers including simple sequence repeat (SSR). In this study a total of 2146 expressed sequence tag (EST)-SSR loci were detected with the 10,737 EST sequences retrieved from the NCBI. A total of 49 EST-SSR markers, including 24 simple ones with a motif comprising of tri-, tetra-, penta-, hexanucleotides, and 25 compound ones, were selected and then primers were designed. Following conventional polymerase chain reaction (PCR) specificity control and sequence authentication, as well as fluorescence-based PCR product size and stutter band evaluation, 37 EST-SSR markers with correct amplification and without stutter band interference were validated. Among them, 14 were polymorphic in 18 closely related peach accessions, with polymorphism information content (PIC) ranging from 0.0994 to 0.3750. The 18 peach accessions can be distinguished using nine polymorphic markers, with the exception of 'Shangshandayulu' and 'Xipu 1', both being bud sports from 'Yulu'. The clustering of the accessions as well as the fingerprint profiles supported the authentication of the hybrids. These EST-SSR markers are useful for peach breeding research.