Abstract: Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F₅ generation plants resulting from inter-subspecific crosses (Nipponbare × KDML105) with low (LS) and high (HS) seed-setting rates, where normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4‒5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes (DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the HS and LS-specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzymes controlling pectin, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase (CYP73), involved in flavonoid biosynthesis, presented higher expression in HS pistils post-pollination. Our findings suggested that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall (OsPME35 and OsPLL9) and controlling later processes like pollen-stigma adhesion (Os02g0467600). Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately leading to the development of high-yielding rice varieties.

Key words: bulked segregant RNA-Seq, flavonoid, inter-subspecific cross, pectin, pollinated pistil, prezygotic barrier, spikelet fertility