Male sterility is an essential trait for minimizing costs and ensuring seed purity in the production of tomato hybrid seeds. However, its application in commercial breeding faces challenges such as instability, low stigma exsertion rates, and the lack of molecular markers to facilitate the efficient backcrossing of male sterile loci into elite tomato lines. This study characterized the male sterile-24 (ms-24) mutant, which demonstrated exserted stigmas and complete male sterility. Our histological examination revealed that ms-24 anthers displayed dysfunctional development of the tetrads and tapetum. The ms-24 locus was finely mapped to a 149 kb interval containing 22 putative genes. Among these, Solyc02g079810, also known as MS-10, encodes a basic helix-loop-helix (bHLH) transcription factor essential for tapetum and pollen development. A sequencing analysis revealed an approximately 4.9 kb retrotransposon insertion in the first exon of the MS-10 gene in ms-24. Allelism tests confirmed that ms-24 was allelic to ms-10, which is also allelic to ms-35 and ms-36. The same retrotransposon insertion was also identified in the ms-10 mutant, and a similar insertion of retrotransposon was detected in the second exon of the MS-10 gene in the ms-36 mutant. Based on these retrotransposon sequences, codominant insertion-deletion (InDel) markers, MS-24I and MS-35I, were developed for the precise identification of the ms-24, ms-10, ms-35, and ms-36 alleles. These findings establish a foundational basis for subsequent investigations into the molecular mechanisms underlying male sterility and enhance the selection process of male sterile lines in tomato hybrid breeding programs.
