Quick creation and mapping of EMS-induced maize kernel mutants identifies classical gene ZmBT1 and novel gene ZmTOP6A

Maize (Zea mays L.) kernel mutants are valuable tools for investigating kernel development. In this study, we employed ethyl methanesulfonate (EMS) mutagenesis of pollen on five inbred lines, which displayed varying performance after treatment. Over 400 independent kernel mutants were generated, showing a wide range of defects in both type and severity. Bulked segregant analysis (BSA) combined with whole-genome sequencing was employed to map two representative mutants. For a shrunken kernel mutant, a missense mutation (P155L) was identified in the classical ZmBT1 gene, which encodes an ADP-glucose transporter. For a small kernel mutant, a start_lost mutation (M1?) was discovered in the ZmTOP6A gene, which encodes the DNA topoisomerase VI subunit A. Allelic verification of ZmBT1 and ZmTOP6A confirmed their association with the mutant phenotypes. Furthermore, we analyzed the protein conservation, expression patterns, and subcellular localization of both genes. Our study highlights the effectiveness of combining EMS mutagenesis with BSA for cloning maize kernel genes. The mutants and the identified genes will advance our understanding of maize kernel development.