Oryza sativa RNA-dependent RNA Polymerase 6 Contributes to Double-strand Break Formation in Meiosis

RNA-dependent RNA polymerase 6 (RDR6) is a core component of the small RNA biogenesis pathway, but its function in meiosis is unclear. Here, we report a new allele of OsRDR6 (Osrdr6-mei), which causes meiosis-specific phenotypes in rice (Oryza sativa). In Osrdr6-mei, meiotic double-strand break (DSB) formation is partially blocked. We created a biallelic mutant with more severe phenotypes, Osrdr6-bi, by crossing Osrdr6-mei with a knockout mutant, Osrdr6-edi. In Osrdr6-bi meiocytes, 24 univalents were observed, and no histone H2AX phosphorylation foci were detected. Compared with the wild type, the number of 21-nt small RNAs in Osrdr6-mei was dramatically lower, while the number of 24-nt small RNAs was significantly higher. Thousands of differentially methylated regions (DMRs) were discovered in Osrdr6-mei, implying that OsRDR6 plays an important role in DNA methylation. There were 457 genes down-regulated in Osrdr6-mei, including three genes, CENTRAL REGION COMPONENT 1 (CRC1), P31comet and Oryza sativa SOLO DANCERS (OsSDS), related to DSB formation. Interestingly, the down-regulated genes were associated with a high level of 24-nt small RNAs, but less strongly associated with DMRs. Therefore, we speculate that the alteration in expression of small RNAs in Osrdr6 mutants leads to the defects in DSB formation during meiosis, which might not be directly dependent on RNA-directed DNA methylation.