Validation of the universality of molecular markers for seedlessness in grape under different genetic backgrounds

【Objective】A universality assessment was conducted on six seedlessness-related molecular markers in grape that have been previously reported both domestically and internationally. This evaluation was performed using a diverse set of 420 grape germplasm accessions to comprehensively verify the effectiveness and reliability of these markers across different genetic backgrounds. The results aim to provide robust technical support for the application of molecular markers in marker-assisted selection (MAS) for the breeding of seedless grape cultivars.【Methods】A total of 420 grapevine accessions were used in the study, comprising a natural population of 68 seedless and 89 seeded accessions, along with two F1 hybrid populations. The first population, derived from the cross of Shine Muscat × Hongyan Seedless, included 134 individuals (35 seedless and 99 seeded), while the second population, from Red Globe × Zhengyan Seedless, included 129 individuals (56 seedless and 73 seeded). Six molecular markers—p1_VvAGL11, p2_VvAGL11, p3_VvAGL11, 5U_VviAGL11, VvSD10, and KASP_VviAGL11—were selected for analysis based on previous studies both domestically and internationally. Polymerase chain reaction (PCR) was used to amplify target fragments. Capillary electrophoresis was applied to determine the allele sizes of SSR markers, while the genotypes of KASP markers were identi-fied using KASP genotyping technology. Chi-square (χ2 ) independence tests (P＜0.05) were performed using R software to assess the association between seedless phenotype and molecular marker alleles and genotypes. The false positive rate, false negative rate, seedlessness identification accuracy, and seedlessness detection rate were calculated for each marker to evaluate their performance and general applicability.【Results】In both F1 hybrid populations, allelic and genotypic analyses of SSR markers showed the following results. p1_VvAGL11: The 250 bp allele showed a highly significant association with seedlessness, while the 257 bp allele was significantly associated with the seeded phenotype. Similarly, the genotype 250/250 was highly associated with the seedlessness phenotype, whereas the genotype 257/ 257 was significantly associated with the seeded phenotype. The 250 bp allele showed seedlessness identification accuracies of 70.90% and 87.60% , with corresponding detection rates of 46.97% and 80.30% in the two hybrid populations, respectively. p2_VvAGL11: The 171 bp allele was moderately significantly associated with seedlessness. The genotypes 158/171 and 171/171 were highly significantly associated with the seedlessness phenotype, while the genotype 158/158 was highly significantly associated with the seeded phenotype. The 171 bp allele showed seedlessness identification accuracies of 63.43% and 64.34% and detection rates of 41.67% and 54.90%. p3_VvAGL11: The 195 bp allele was highly significantly associated with seedlessness. The genotype 185/195 was highly significantly associated with seedlessness, while the genotype 185/185 was highly significantly associated with the seeded phenotype. The 195 bp allele showed seedlessness identification accuracies of 74.63% and 89.92% and detection rates of 50.82% and 85.25%. 5U_VviAGL11: The 316 bp allele was highly significantly associated with the seedlessness phenotype, while the 308 bp and 318 bp alleles were highly significantly associated with the seeded phenotype. the genotypes 304/316 and 308/316 were highly associated with the seedlessness phenotype, while the genotypes 304/308, 304/318, and 308/318 were strongly associated with the seeded phenotype. The 316 bp allele showed seedlessness identification accuracies of 73.13% and 89.15% and detection rates of 49.21% and 85.00%. VvSD10: The 105 bp allele was significantly associated with the seedlessness phenotype, while the 113 bp allele was highly associated with the seeded phenotype. Genotype 105/107 was highly associated with the seedlessness phenotype, while genotypes 107/107 and 107/113 were strongly associated with the seeded phenotype. The 105 bp allele showed seedlessness identification accuracies of 76.12% and 91.47% , with corresponding detection rates of 52.54% and 88.14% , respectively. Among germplasm carrying the corresponding alleles, the proportion of seedless individuals was 63.6% for p1_VvAGL11, 48.9% for p2_VvAGL11, 68.0% for p3_VvAGL11, 66.7% for 5U_VviAGL11, and 70.3% for VvSD10. Among germplasm carrying the corresponding genotypes, the proportions were 69.8%, 63.2%, 80.1%, 72.6%, and 84.0%, respectively. The KASP_VviAGL11 marker allele A was significantly associated with seedlessness. The genotypes A:A and A:C corresponded to the seedlessness phenotype, whereas genotype C:C corresponded to the seeded phenotype. Both false positive and false negative rates were observed in the two hybrid populations. The A allele showed seedlessness identification accuracies of 75.37% and 89.92% , detection rates of 51.67% and 85.25% , respectively. In the natural population, the A allele of KASP_VviAGL11 was 100% associated with the seedless phenotype in diploid germplasm.【Conclusion】Six seedlessness-related molecular markers were validated in the two F1 hybrid populations of Shine Muscat × Hongyan Seedless and Red Globe×Zhengyan Seedless. Among them, VvSD10 and KASP_VviAGL11 exhibited the highest seedlessness identification accuracies and detection rates, along with the lowest false positive and false negative rates, thus showing the best overall performance. The 5U_VviAGL11 marker also demonstrated high accuracy and detection rates, low error rates, and provided rich genetic informa-tion. In contrast, p2_VvAGL11 had the lowest accuracy and detection rate. These results indicate that certain molecular markers, particularly VvSD10 and KASP_VviAGL11, have strong potential for use in marker-assisted selection in seedless grape breeding.