Development of a reproductive calendar in Eragrostis curvula to guide transcriptomic studies of apomixis

Abstract

Eragrostis curvula (weeping lovegrass) is a model system for studying diplosporous apomixis due to its unique reproductive mode, wide range of ploidy levels and reproductive behaviors and the availability of genomic and epigenomic tools. A major limitation for transcriptomic studies in this species has been the difficulty to isolating female reproductive tissues at precise developmental stages, without other floral tissues that introduce expression noise from non-target tissues. To overcome this, we performed a detailed cytoembryological and morphometric characterization of male and female gametophyte development in seven E. curvula genotypes with distinct ploidies and reproductive modes (sexual, facultative apomictic, and obligate apomictic) to identify relationships between floral parameters and female reproductive developmental stages. Using differentialinterference contrast (DIC) microscopy and methyl salicylate clarification, we identified cytological markers that distinguish apomictic and sexual female reproductive developmental stages. These stages were then correlated with external floral parameters, such as pistil and anther length, to establish a genotype-specific reproductive calendar. Among the measured traits, pistil length showed the highest consistency with developmental stages, enabling accurate staging of ovules—particularly for early stages critical to cell fate determination. Synchrony between male and female reproductive development was also evaluated, and no consistent differences were observed across genotypes with different ploidy levels or reproductive modes. RNA extraction from pistils atselected reproductive developmental stages yielded high-quality mRNA suitable for transcriptomic analysis. Our approach enhances the resolution and accuracy of developmental stage-specific sampling, and it lays the foundation for dissecting the genetic basis of apomixis in E. curvula, with potential applications for engineering clonal reproduction in crop species.