Email: thien.tran@research.uwa.edu.au

Plant mitochondrial genomes may carry genes causing cytoplasmic male sterility (CMS), resulting in failed pollen development and seed set. Proteins encoded by nuclear Restorer-of-fertility (Rf) genes can block CMS gene expression, forming an effective breeding system (CMS/Rf) that controls self-pollination and restores fertility in F1 hybrids.

Although hybrid varieties have been developed successfully in various crops, wheat lacks an efficient breeding system, hindering hybrid seed production on a commercial scale. A promising system in wheat involves the mitochondrial orf279 gene in Triticum timopheevii (T-CMS) and nuclear Rf1 and Rf3 genes. It has been shown that Rf1 and Rf3 proteins target the orf279 transcript and induce cleavage to suppress the T-CMS phenotype.

However, seed-set experiments indicated neither of the two restorer genes can fully restore the fertility of F1 hybrids, a crucial requirement of a successful breeding system, contrasting with fully fertile T. timopheevii plants.

Using next-generation sequencing assays and PCR-based sequence analyses, we compared the expression and processing of orf279 in T. timopheevii with transgenic wheat plants. We observed that orf279 is more suppressed in T. timopheevii than transgenic wheat plants carrying T-CMS and single Rf1 or Rf3 or both genes. RNA-seq analysis indicated that the low expression levels of Rf genes may be insufficient to block the expression of orf279 in transgenic lines.

Post-transcriptional gene silencing mechanisms have been reported to affect Rf gene expression in plants negatively. Our small RNA-seq and degradome-seq data analyses revealed several small RNAs targeting and cleaving Rf transcripts. To explore the extent to which the expression of Rf genes is regulated by those small RNAs, we investigated accessions with targeted changes on the small RNA binding sites.

Our comprehensive analysis sheds new light on the possible reasons for inconsistent fertility restoration using T-CMS/Rf systems, offering valuable insights for high-yielding hybrid wheat production.