Direct fluorescent S-genotyping reveals genetic diversity and pedigree inconsistencies in red-fleshed apple hybrids and American heritage varieties.

Main conclusion A PCR-only S-genotyping was optimized for red-fleshed apple hybrids carrying the R6 allele of MdMYB10. Sixteen distinct S-alleles were identified. New hybrids show unique S-allele frequencies, enhancing breeding diversity. Red-fleshed apples are increasingly valued for their esthetic appeal and potential health benefits, making them attractive targets in modern breeding programs. In this study, 80 novel red-fleshed and six white-fleshed apple hybrids, along with their parental cultivars, were genotyped for the R6 allele of the MdMYB10 gene and for their S-alleles to assess pollination compatibility. All red-fleshed hybrids carried the R6 allele, confirming their type I red-fleshed phenotype. A previously published high-throughput S-genotyping protocol was optimized to achieve single base-pair accuracy, enabling a streamlined, PCR-only workflow without the need for restriction enzyme digestion. 16 distinct S-alleles were identified, and allele-specific primers and sequencing were used to verify ambiguous cases. The genotyping results revealed inconsistencies in pedigree records and pollination procedures, underscoring the importance of molecular validation in breeding programs. Additionally, two American heritage apple cultivars were found to carry three S-alleles despite being diploid, suggesting possible segmental duplications. The S-allele frequency distribution in the new hybrids differed from traditional Hungarian and international germplasm, indicating their potential to broaden the genetic base of disease-resistant apple breeding. This study provides a refined genotyping approach and valuable insights into the genetic composition of novel apple hybrids, contributing to improved breeding strategies and germplasm management.