Centromeres are specified by the histone variant CENH3, which can interact with centromeric retroelements and tandem repeat arrays to determine the locations and sizes of active centromeres. Centromere mediated haploid induction occurs when crossing wild-type plants with plants expressing CENH3 variants. Centromere size variation has been proposed to underlie uniparental genome elimination that leads to haploid formation. In this hypothesis, the centromere size dimorphism between the wild type and CENH3 variant genomes in the zygote causes the removal of the smaller centromeres and uniparental genome elimination. In this study, we employed multiple approaches to explore centromere size and its impact on haploid induction in maize. We first investigated the centromere size across 26 maize inbred lines and demonstrated that centromere size is not influenced by centromere sequence but positively correlated with chromosome size and genome size. We then confirmed the relationship between genome size and centromere size by introducing maize centromeres into two larger genome backgrounds Oaxaca and Zea luxurians. Our results suggest that maize centromeres are expanded in the larger genome background. Literature from other species showed that overexpression of CENH3 could cause the ectopic formation of functional centromeres. However, we found threefold overexpression of CENH3 in maize did not significantly increase centromere size. Taken together, these results suggest that centromere size is scalable with genome size and controlled by multiple limiting factors but not exclusively to CENH3.

To test the centromere size model for haploid induction, we utilized CRISPR/Cas9 to create a maize cenh3 null mutant that is homozygous lethal but poorly transmissible through both male and female gametophytes. We found that haploids were formed at high frequency when wild-type plants are crossed with cenh3 heterozygous mutants either as female (haploid induction ratio 5%) or male (0.5%). Our results are consistent with the hypothesis that diminished/small centromeres induce haploid formation. Genotyping endosperm of the seeds that gave rise to haploid plants showed that all were fertilized by cenh3 pollen. Since CENH3 is present in all plants, this method may have potential to induce haploid across diverse species.