Comprehensive analysis of imprinted genes in citrus endosperm and their contributions to seed development

Interploidy hybridization between diploid and tetraploid has been an important approach to develop triploid hybrids in horticultural plants including citrus for seedless breeding. However, dysregulation of imprinted genes can lead to the failure of endosperm cellularization, resulting in abortion of triploid embryos before seed maturity, thereby impeding the efficiency of triploid generation. The identification of imprinted genes is essential for comprehending the impact of imprinting on endosperm cellularization and mitigating embryo abortion in interploidy hybridization. Herein, a genome-wide search for imprinted genes in citrus was performed using RNA sequencing of the endosperm collected from two pairs of reciprocal crosses and totally 296 imprinted genes were identified. Among them, the paternally imprinted genes (PEGs) showed a higher inclination toward endosperm-specific expression compared to maternally imprinted genes (MEGs), with a few demonstrating consistent imprinting across three developmental stages of endosperm and displaying homology with counterparts found in other plant species. The analysis of transposable element (TE) enrichment and DNA methylation revealed a significant enrichment of mutator TEs with higher level of DNA methylation around maternal imprinted genes, highlighting their vital role for controlling the expression of MEGs. By overexpressing the endosperm-specific expressed PEGs in Arabidopsis, we observed that the transgenic lines of OE-CsPEG1 exhibited an increased frequency of seed abortion, which appeared to be correlated with delayed endosperm cellularization, resembling the observed phenotypes in 2x × 4x interploidy hybridizations. Our research provides evidence supporting the functional conservation of imprinted genes in plants, thereby identifying potential targets for genetic improvement in triploid breeding.