Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T and F plants, and genotyping and phenotyping of F progenies, we further confirmed gene functions of every member in family, and identified the functions of , , , and in controlling maize male fertility. Single and double homozygous gene mutants of did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the single gene was required for anther and pollen development, while had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.