Discovery of genes involved in maize cytoplasmic male sterility (CMS) fertility restoration

Abstract

Commercial hybrid corn production provides high yielding seed corn; however, it requires detasseling of female parent plants within the production fields or an alternate mode of tassel sterility. Cytoplasmic Male Sterility (CMS) is a maternally inherited trait caused by chimeric open reading frames in mitochondrial DNA. Female plants with CMS cytoplasm do not produce functional pollen and do not need to be manually or mechanically detasseled. Nuclear restoration of fertility (Rf) genes carried by the male parent plants reduce the effect of the chimeric mitochondrial gene products and restore functional pollen in the hybrid plants. Two systems of CMS are used in commercial hybrid corn production, CMS-C and CMS-S. CMS-C is more widely used in the industry than CMS-S because CMS-C is more environmentally stable. The major restoring locus for CMS-C is Rf4 and for CMS-S is Rf3, although both systems have additional partial restoring loci. This study aims to explore the gene networks responsible for CMS-C and CMS-S fertility restoration. My first research project investigated the functional allele of Rf4 associated with CMS-C restoration. The Rf4 gene was fine mapped to a small map based cloning interval containing a transcription factor. I predicted the Y187F substitution was associated with restoring ability and then created a CRISPR-edited isoline for this variant. This variant showed the restoring trait was associated with the amino acid substitution. Using structural modeling, I predicted this Y187F substitution was in a critical hetero-dimer interface. I calculated the allele frequency of the restoring allele among heterotic groups in a diversity panel and determined the restoring allele was in 64% of Non-Stiff Stalk material and the non-restoring allele was in 83% of Stiff Stalk material. Through my study of Rf4 alleles, I discovered that some inbreds contained the restoring Rf4 allele yet were incapable of restoration. My second research project sought to explore the genetic cause of this failure to restore phenotype. Through QTL mapping, I found the trait mapped to a PPR gene cluster on chromosome 2. Through fine-mapping and genomic analysis, I identified three candidate genes that I used for transgenic complementation tests. The complementation tests showed the causal gene, PPR153, is involved in Rf4 restoration; however, PPR153 is unable to restore CMS-C independent of Rf4. My final research project aimed to identify the causal Rf3 gene associated with CMS-S restoration. I used fine-mapping and genomic analysis to identify two candidate genes for Rf3. Using gene complementation tests of the candidate genes, I determined PPRK2 had partial restoration but was unable to generate viable pollen. Using RT-PCR for the sterility inducing factor, I showed PPRK2 reduced the transcript amount. These three research projects together improved our understanding of maize CMS/Rf systems and allow for increased usage of CMS in hybrid seed production.