Male sterility is used to mass-produce F₁ hybrid seeds in pepper (Capsicum annuum L.). In particular, the development of genetic male sterility (GMS)-linked molecular markers may play a crucial role in hybrid breeding of pepper. To date, approximately 20 GMS genes have been identified in pepper. Among these, several molecular markers for the ms₃ gene have been developed in previous studies; however, they are not completely linked and thus have limitations for use in selection. Therefore, in the present study, we aimed to develop molecular markers for ms₃ selection using single-nucleotide polymorphism (SNP)-based high-resolution melting (HRM) analysis. Chi-square test was conducted using three F₂ segregating populations, and the results confirmed a 3:1 segregation ratio between male-fertile and male-sterile plants. A total of 128 primer sets were designed by selecting SNPs near the ms₃ gene, and 25 HRM markers were successfully developed. Using 420 individuals from the F₂ segregating population ‘GMS3,’ a high-density genetic linkage map of pepper chromosome 1 was constructed, with eight HRM markers found to be co-segregated with the ms₃ gene. Subsequent experiments using various plant materials validated these eight markers, ultimately identifying two HRM markers, HRM119655681 and HRM135273656, for the final selection. These two markers showed co-segregation between the phenotype and genotype of ms₃ across all plant materials used in the study. The markers developed in this study are expected to be effective for maternal line development and large-scale F₁ hybrid seed production using ms₃ in pepper.

In this study, genotype analysis was conducted using the Fluidigm system with 13 SNP markers related to anthracnose, powdery mildew, phytophthora root rot, cucumber mosaic virus (CMV), potyvirus, and tobacco mosaic virus (TMV) resistance. A total of 25,350 data points were obtained, 8.2% of which indicated resistance genotypes. Resources having perfect resistance genotype to each disease-related molecular marker set were 58 accessions for anthracnose, 57 for powdery mildew, 246 for phytophthora root rot, and 765 for CMV. There was no resource having a perfect resistance genotype of all three potyvirus-related molecular SNP markers including pvr1, pvr2 (123457), and pvr2 (689). Thirty-seven accessions for pvr1, 349 accessions for pvr2 (123457), and 23 accessions for pvr2 (689) were identified as resources with resistance genotype. No resource with the TMV-resistance genotype was found in this study. The resources with complex resistance genotypes of anthracnose and powdery mildew were 55 accessions, and they were mainly Capsicum baccatum. Among these, seven accessions had a three-disease combined resistance genotype with phytophthora root rot. Thirty-six accessions, 16 accessions, and nine accessions each had resistance genotypes of the three potyvirus-related markers. These resources also had CMV and phytophthora root rot resistance genotypes, and they were mainly Capsicum chinense. The statistical analysis considering the evaluated resistance genotypes, revealed that the anthracnose resistance genotype showed a tendency to be linked with powdery mildew resistance genotype. The potyvirus-related marker pvr1 was positively correlated with Phytophthora root rot markers.