Phasing analysis of the transcriptome and epigenome in a rice hybrid reveals the inheritance and difference in DNA methylation and allelic transcription regulation.

Hybrids are always a focus of botanical research and have a high practical value in agricultural production. To better understand allele regulation and differences in DNA methylation in hybrids, we developed a phasing pipeline for hybrid rice based on two parental genomes (PP2PG), which is applicable for Iso-Seq, RNA-Seq, and Bisulfite sequencing (BS-Seq). Using PP2PG, we analyzed differences in gene transcription, alternative splicing, and DNA methylation in an allele-specific manner between parents and progeny or different progeny alleles. The phasing of Iso-Seq data provided a great advantage in separating the whole gene structure and producing a significantly higher separation ratio than RNA-Seq. The interaction of hybrid alleles was studied by constructing an allele co-expression network that revealed the dominant allele effect in the network. The expression variation between parents and the parental alleles in progeny showed tissue- or environment-specific patterns, which implied a preference for trans-acting regulation under different conditions. In addition, by comparing allele-specific DNA methylation, we found that CG methylation was more likely to be inherited than CHG and CHH methylation, and its enrichment in genic regions was connected to gene structure. In addition to an effective phasing pipeline, we also identified differentiation in OsWAK38 gene structure that may have led to the expansion of allele functions in hybrids. In summary, we developed a phasing pipeline and provided valuable insights into alternative splicing, interaction networks, trans-acting regulation, and the inheritance of DNA methylation in hybrid rice.