This proposal describes a next-generation sequencing (NGS)-based approach to identify genes that control unisexual flower development in Zea mays (maize). Maize develops separate male flowers in the tassel and female flowers in the ear (Klein etal., 2018). The development of unisexual flowers is important for hybrid crop production - separate tassel and ear flowers allow humans to very easily make controlled crosses (Phillips, 2010). Many cereal crops related to maize, like rice and wheat, have unisexual flowers, hampering hybrid seed production (Kellogg, 2015). In addition, the same process that leads to the development of maize flowers in the tassel - carpel suppression - also occurs in half of all ear flowers, effectively halving the number of seeds a maize ear could produce (Cheng et al., 1983). Thus, modifying the genes that control carpel suppression using CRISPR/Cas9 genome engineering could allow crop engineers to generate unisexual flowers in other grass crops, and to improve yield in maize (Gao, 2018). However, the genes that specifically control carpel suppression remain poorly understood. Here, we propose to almost double the number of genes with potential roles in carpel suppression using an NGS-based protocol for mapping genes to chromosomal locations and identifying candidate genes (Klein et al., 2018). Until recently, the process of cloning maize genes was arduous and time-consuming (Gallavotti and Whipple, 2015). The rise of next-generation sequencing technology, as well as a fully sequenced maize genome, now makes it possible to quickly map genes to a chromosomal location and identify gene candidates using bulked segregant analysis coupled to high throughput sequencing (BSA-Seq) (Klein et al., 2018; Michelmore et al., 1991). In a BSA-Seq experiment, a mutant of interest is crossed to a wild-type individual in a contrasting genetic background. Heterozygous F1 individuals are then selfed or backcrossed, and mutants with recombinant chromosomes are identified in the resulting F2 population. These mutants can then be used to identify a region of increased homozygosity in chromosomal regions physically linked to causative lesions . DNA from a pool of these mutants is then sequenced (Fig. 1). Downstream analyses identify thousands of polymorphisms that differ between the two parental genotypes, and detect genotypes that are over-represented (i.e. linked to causative lesions) in the mutant segregant pool. Once a mapping interval has been identified, the sequencing data can be examined for potentially causative lesions. Thus, BSA-Seq allows researchers to identify small mapping intervals--and often causative lesions--relatively quickly (Klein et al., 2018). Here, we will use BSA-Seq to map seven genes with roles in carpel suppression: two mutants that arose from an EMS mutagenesis screen in our lab, four mutants from the Maize Genetics Cooperative Stock Center (referred to hereafter as the "Co-op"), and a naturally occurring modifier of carpel suppression from the P39 genetic background. Previous Hatch funding was sufficient to cover some nursery expenses and some whole genome shotgun sequencing runs. Therefore, I have limited this proposal to the field work, mutant characterization, and initial sequencing necessary to map the P39 modifier and the six focal mutant genes.
