Fine-Mapping of Tomato <i>male sterile-24</i> Locus and Marker Development for <i>ms-24</i> and Its Alleles, <i>ms-10</i>, <i>ms-35</i>, and <i>ms-36</i>

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 <i>male sterile-24</i> (<i>ms-24</i>) mutant, which demonstrated exserted stigmas and complete male sterility. Our histological examination revealed that <i>ms-24</i> anthers displayed dysfunctional development of the tetrads and tapetum. The <i>ms-24</i> locus was finely mapped to a 149 kb interval containing 22 putative genes. Among these, <i>Solyc02g079810</i>, also known as <i>MS-10</i>, 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 <i>MS-10</i> gene in <i>ms-24</i>. Allelism tests confirmed that <i>ms-24</i> was allelic to <i>ms-10</i>, which is also allelic to <i>ms-35</i> and <i>ms-36</i>. The same retrotransposon insertion was also identified in the <i>ms-10</i> mutant, and a similar insertion of retrotransposon was detected in the second exon of the <i>MS-10</i> gene in the <i>ms-36</i> mutant. Based on these retrotransposon sequences, codominant insertion–deletion (InDel) markers, MS-24I and MS-35I, were developed for the precise identification of the <i>ms-24</i>, <i>ms-10</i>, <i>ms-35</i>, and <i>ms-36</i> 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.