Background: Grapevine breeding increasingly relies on molecular tools to introduce durable resistance to downy and powdery mildew. However, the reproducibility of simple sequence repeat (SSR) markers across platforms remains a challenge for marker-assisted selection (MAS). This study aimed to evaluate the performance of SSR markers associated with key resistance loci (Run1/Rpv1, Ren3/Ren9, Rpv3, Rpv10, Rpv12) using the Qsep100 system and to validate selected markers on the ABI platform. Methods: A panel of grapevine cultivars and breeding genotypes was analyzed for SSR markers linked to resistance loci. PCR amplicons were separated on the Qsep100 BioFragment Analyzer, and a subset of markers was cross-validated using ABI capillary electrophoresis. Results: Only a limited subset of markers displayed consistent performance across genotypes. Sc34-8 and Sc35-2 were most reliable for Run1/Rpv1, Indel-27 and Indel-20 for Ren3/Ren9, UDV737 for all Rpv3 sub-loci, GF-09-44 and GF-09-57 for Rpv10, and UDV340 and UDV343 for Rpv12. ABI validation of UDV737 and Indel-27 confirmed high concordance with Qsep100 results, with allele size differences typically ≤2 bp. Conclusions: The study identifies a core set of robust SSR markers suitable for routine MAS in grapevine breeding. Results demonstrate that the Qsep100 system is a reliable alternative to ABI for large-scale genotyping, supporting its broader implementation in resistance breeding programs.
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