Candidate Gene Identification and Transcriptome Analysis of Tomato male sterile - 30 and Functional Marker Development for ms - 30 and Its Alleles, ms - 33 , 7B - 1 , and stamenless - 2 .

Male sterility is a valuable trait for hybrid seed production in tomato ( Solanum lycopersicum ). The mutants male sterile - 30 ( ms - 30 ) and ms - 33 of tomato exhibit twisted stamens, exposed stigmas, and complete male sterility, thus holding potential for application in hybrid seed production. In this study, the ms - 30 and ms - 33 loci were fine-mapped to 53.3 kb and 111.2 kb intervals, respectively. Tomato PISTILLATA ( TPI , syn. SlGLO2 ), a B-class MADS-box transcription factor gene, was identified as the most likely candidate gene for both loci. TPI is also the candidate gene of tomato male sterile mutant 7B - 1 and sl - 2 . Allelism tests revealed that ms - 30 , ms - 33 , 7B - 1 , and sl - 2 were allelic. Sequencing analysis showed sequence alterations in the TPI gene in all these mutants, with ms - 30 exhibiting a transversion (G to T) that resulted in a missense mutation (S to I); ms - 33 showing a transition (A to T) that led to alternative splicing, resulting in a loss of 46 amino acids in protein; and 7B - 1 and sl - 2 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 TPI gene-edited mutants and allelism tests indicated that the gene TPI is responsible for ms - 30 and its alleles. Transcriptome analysis of ms - 30 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 ms - 30 and its alleles in tomato breeding and support the functional analysis of the TPI gene.