Male-sterile lines constitute a principal means of exploiting heterosis and advancing genetic breeding in pepper (Capsicum annuum L.). Fine mapping of the Genic male sterility (GMS) genes can provide relevant gene resources for subsequent gene cloning, functional verification, molecular mechanism analysis, and creation of new germplasm, laying the foundation for the development of nuclear male sterile lines in pepper.

A natural GMS mutant pby-1 and its wild type PBY-1 were used as parents for hybridization to obtain F₁ progeny, which were then self-pollinated to F₂ generation. Thirty plants showing sterile and normal phenotypes were selected from the F₂ population to construct two extreme pools. Through bulked segregant analysis sequencing (BSA-Seq), candidate regions associated with male sterility were identified, and candidate genes within the regions were mined. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were conducted. Further, the candidate genes were jointly analyzed with the differentially expressed proteins of the parents pby-1 and PBY-1 at different developmental stages to narrow down the range of candidate genes related to nuclear male sterility in pepper. The expression levels of the candidate genes in pepper flower organs were detected by real-time fluorescence quantitative PCR to verify their potential in regulating male sterility in pepper.

Through BSA-Seq sequencing analysis, 12 regions associated with male sterility were identified on chromosome Chr.07, with a total length of approximately 70.02 Mb, containing 343 candidate genes, which did not overlap with the known ms-1, ms-2, and ms-3 sterility gene regions. The results of GO enrichment and KEGG enrichment analysis indicated that carbohydrate metabolism, lipid transport metabolism, and plant hormone signal transduction pathways were closely related to male sterility in pepper. Through comprehensive analysis combining proteomics data, 12 candidate genes (Capana07g000676, Capana07g000956, Capana07g000979, Capana07g000993, Capana07g001228, Capana07g001239, Capana07g001241, Capana07g001254, Capana07g001294, Capana07g001295, Capana07g001312, and Capana07g001315) were screened. Through qRT-PCR detection, Capana07g000676, Capana07g000979, Capana07g000993, Capana07g001228, Capana07g001241, and Capana07g001294 were identified as key candidate genes for male sterility.

By combining BSA-Seq with proteomics, the functional region of nuclear male sterility was located on chromosome 7 of the pepper, and six core candidate genes were screened.