Candidate Gene Identification and Transcriptome Analysis of Tomato - and Functional Marker Development for - and Its Alleles, -, -, and -.

Male sterility is a valuable trait for hybrid seed production in tomato (). The mutants - (-) and - of tomato exhibit twisted stamens, exposed stigmas, and complete male sterility, thus holding potential for application in hybrid seed production. In this study, the - and - loci were fine-mapped to 53.3 kb and 111.2 kb intervals, respectively. (, syn. ), a B-class MADS-box transcription factor gene, was identified as the most likely candidate gene for both loci. is also the candidate gene of tomato male sterile mutant - and -. Allelism tests revealed that -, -, -, and - were allelic. Sequencing analysis showed sequence alterations in the gene in all these mutants, with - exhibiting a transversion (G to T) that resulted in a missense mutation (S to I); - showing a transition (A to T) that led to alternative splicing, resulting in a loss of 46 amino acids in protein; and - and - mutants showing the insertion of an approximately 4.8 kb retrotransposon. On the basis of these sequence alterations, a Kompetitive Allele Specific PCR marker, a sequencing marker, and an Insertion/Deletion marker were developed. Phenotypic analysis of the gene-edited mutants and allelism tests indicated that the gene is responsible for - and its alleles. Transcriptome analysis of - and quantitative RT-PCR revealed some differentially expressed genes associated with stamen and carpel development. These findings will aid in the marker-assisted selection for - and its alleles in tomato breeding and support the functional analysis of the gene.