Features of sRNA biogenesis in rice revealed by genetic dissection of sRNA expression level

We previously conducted a QTL analysis of small RNA (sRNA) abundance in flag leaves of an immortalized rice F₂ (IMF2) population by aligning sRNA reads to the reference genome to quantify the expression levels of sRNAs. However, this approach missed about half of the sRNAs as only 50% of all sRNA reads could be uniquely aligned to the reference genome. Here, we quantified the expression levels of sRNAs and sRNA clusters without the use of a reference genome. QTL analysis of the expression levels of sRNAs and sRNA clusters confirmed the feasibility of this approach. sRNAs and sRNA clusters with identified QTLs were then aligned to the high-quality parental genomes of the IMF2 population to resolve the identified QTLs into local vs. distant regulation mode. We were able to detect new QTL hotspots by considering sRNAs aligned to multiple positions of the parental genomes and sRNAs unaligned to the parental genomes. We found that several local-QTL hotspots were caused by sequence variations in long inverted repeats, which probably function as precursors of sRNAs, between the two parental genomes. The expression levels of these sRNAs were significantly associated with the presence/absence of the long inverted repeats in the IMF2 population. Moreover, we found that the variations in whole-genome sRNA species composition among different IMF2s were attributed to sRNA biogenesis genes including OsDCL2b and OsRDR2. Our results highlight that genetic dissection of sRNA expression is a promising approach to disclose new components functioning in sRNA biogenesis and new mechanisms of sRNA biogenesis.