The Combination of Start-Codon-Targeted (SCoT) and Falling Stone (FaSt) Transposon-Specific Primers Provides an Efficient Marker Strategy for Prunus Species

A novel primer (FaSt-R) targeting the <i>Prunus</i>-specific <i>Falling Stone</i> (<i>FaSt</i>) non-autonomous transposon was combined with start-codon-targeted (SCoT) primers to assess genetic diversity in 12 cultivars from six <i>Prunus</i> species and 28 cultivars of European plum. Compared to SCoT-only analyses, the SCoT-FaSt combination produced fewer total bands but a higher percentage of polymorphic bands, while maintaining comparable values for polymorphism information content, resolving power, gene diversity, and Shannon's index. SCoT-FaSt markers generated bands across a broader size range, which made gel patterns less dense, enabling the more accurate detection of differentially amplified fragments. Neighbor-joining and principal component analyses confirmed that SCoT-FaSt markers provided sufficient phylogenetic resolution at both interspecific and intraspecific levels. The sequencing of 32 SCoT-FaSt amplicons revealed <i>FaSt</i> elements in 26 fragments, with SCoT primers preferentially annealing to GC-rich exonic and intergenic regions. Seventeen protein-coding and one RNA-coding gene were partially identified, with <i>FaSt</i> elements localized in UTRs and introns of genes with key physiological functions. Comparative analysis indicated a biased distribution of <i>FaSt</i> elements between the <i>Cerasus</i> and <i>Prunus</i> subgenera. In silico findings suggest that <i>FaSt</i> elements are more fragmented in cherry species, potentially contributing to subgeneric divergence. Overall, the SCoT-FaSt marker system is effective for evaluating <i>Prunus</i> genetic diversity, reconstructing phylogenetic relationships, and elucidating the genomic impact of an active Mutator-like transposon.