Integrative gene expression and heterologous functional analysis identify candidate regulators of apomixis in...

Eragrostis curvula (weeping lovegrass) is a perennial forage grass in which diploid genotypes reproduce sexually whereas polyploids display pseudogamous diplosporous apomixis. Understanding the molecular basis of this reproductive system is important for the potential use of apomixis in crop improvement. Here, we applied a comparative gene expression analysis to identify and functionally assess candidate genes associated with apomictic reproduction in E. curvula.

This analysis was performed using a custom 1×1M Agilent microarray designed from floral transcriptomes of sexual and apomictic genotypes. PCR screening across 14 genotypes and a segregating mapping population was conducted to evaluate candidate gene presence. Functional assessment was carried out through heterologous expression of the candidate genes in Arabidopsis thaliana using GoldenBraid 2.0 expression cassettes.

Of the 131 differentially hybridized probes identified, 130 were upregulated in apomictic plants. BLAST analyses revealed four main candidate genes, including a hypothetical protein (EcAPO1), a pre-mRNA splicing factor CWC22-like homolog (EcAPO2), a Cyclin-2A-1 (EcAPO3), and an F-box protein (EcAPO4). PCR screening showed that EcAPO1 and EcAPO2 were exclusively present in apomictic individuals, supporting their close association with the trait. Transgenic lines displayed abnormal floral and reproductive phenotypes, including homeotic transformations, supernumerary nuclei, embryo sac arrest, and altered endosperm development.

These results indicate that E. curvula candidate genes can disrupt conserved reproductive pathways and demonstrate biological activity in a heterologous system. Together, this integrative analysis combining comparative gene expression profiling and functional assays identifies novel candidate genes associated with apomixis in E. curvula. These findings provide a foundation for dissecting the genetic control of apomixis and advance efforts toward the applied manipulation of clonal seed reproduction in crops.