Spatial Transcriptomic Profiling Reveals Candidate Genes Associated with Male Gametophyte Development in CMS-D8...

Gossypium trilobum cytoplasmic male sterility (CMS-D8) is characterized as being of the gametophytic type. Meiosis in F1 hybrids leads to the production of both fertile and sterile male gametophytes, making them a suitable material for studying the development of male gametophytes. Paraffin section analysis indicated that stage 8 represents a critical developmental period associated with male gametophyte abortion. Spatial transcriptomic analysis of 4.5-5mm F1 buds revealed distinct spatial expression patterns, including enrichment of genes related to energy production and conversion in a putative meiotic-cell-associated spatial cluster. Cell-level analysis further resolved two transcriptionally distinct male gametophyte populations, between which genes associated with mitochondrial structure and cellular metabolic processes were differentially expressed. GhAAT showed differential expression between these two male gametophyte populations. Furthermore, phenotypic analysis of the Arabidopsis aat mutant revealed reduced pollen viability and a lower proportion of fully developed seeds, providing cross-species evidence consistent with a possible involvement of AAT in male reproductive development. In summary, this study provides a spatial transcriptomic profile of D8F1 buds and identifies GhAAT as a candidate gene associated with CMS-D8 pollen formation. These findings provide candidate genes and spatial information for further investigation of male gametophyte development in CMS-D8 cotton.