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

Abstract

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.

Keywords: QTL mapping; Rice; Small RNA; sRNA; sRNA biogenesis; sRNA expression level.

Graphical abstract

Fig. 1

The distribution of sQTLs and scQTLs across the 1,567 bins. The top four panels are QTLs for sRNAs of 21, 22, 23 and 24 nt while the panel at the bottom shows the QTLs for sc-traits. The 1,567 bins are represented as black bars and are arranged from left to right based on their genomic positions. The width of each bar represents the size of the bin. The chromosome identifiers are labeled on the X-axis. Adjacent chromosomes are represented by different colors. Bins harboring sRNA biogenesis genes are indicated with red rectangles above the bins. Clusters of QTL regulating the expression of large numbers of sRNAs are indicated with blue rectangles above the bins. The bin IDs and the sRNA biogenesis genes for representative bins are indicated in the uppermost panel. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Diverse features of uniquely mapped sRNAs regulated by different sQTL hotspots. sRNAs regulated by each sQTL hotspot are categorized into different groups based on varying features of sRNAs, including the size of sRNAs (A), the genomic distribution of sRNAs (B), the genic distribution of sRNAs (C), the nucleotide preference at the first base position at the 5′ end (D), the origination of sRNAs from MITEs (E). Different sQTL hotspots are represented by different colors (F). All sRNAs with detected QTL are also grouped based on diverse feature of sRNAs and are labeled with grey color. The species of sRNAs in different groups are compared with that of all sRNAs with QTL using chi-squared test. *, p-value < 1e-5. **, p-value < 1e-10.

Fig. 3

sRNAs regulated by Bin795 and the long inverted repeat functioning as the potential precursors of these sRNAs. (A) Expression values of sRNAs regulated by Bin795 in Zhenshan 97 (ZS97), Minghui 63 (MH63) and the hybrid. (B) Expression values of sRNAs regulated by Bin795 in IMF2s of different genotypes. ZS97, the Zhenshan 97 genotype. MH63, the Minghui 63 genotype. Heterozygote, heterozygote genotype. (C) Structure of the long inverted repeat (LIR) (chr06:1989522–2005555) in the Zhenshan 97 genome created using shinyCircos . The clockwise grey circle indicates the LIR. The two complementary regions are connected by the blue ribbon. (D) Structure of the LIR (chr06:1037057–1054781) in the Minghui 63 genome. (E) Structure of the LIR (chr06:1185416–1203145) in the Nipponbare genome. (F) Expression values of the LIR in IMF2s of different genotypes quantified by mRNA sequencing. (G) Correlation coefficients between expression values of sRNAs regulated by Bin795 and the LIR in IMF2s of Zhenshan 97 genotype. (H) Correlation coefficients between expression values of sRNAs regulated by Bin795 and the LIR in IMF2s of Minghui 63 genotype. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

sRNAs regulated by Bin1556 and the long inverted repeat functioning as the potential precursors of these sRNAs. (A) Expression values of sRNAs regulated by Bin1556 in Zhenshan 97 (ZS97), Minghui 63 (MH63) and the hybrid. (B) Expression values of sRNAs regulated by Bin1556 in IMF2s of different genotypes. ZS97, the Zhenshan 97 genotype. MH63, the Minghui 63 genotype. Heterozygote, heterozygote genotype. (C) Structure of the long inverted repeat (LIR) (chr12:22977717–22980753) in the Minghui 63 genome. The clockwise grey circle indicates the LIR. The two complementary regions are connected by the blue ribbon. (D) Structure of the LIR (chr12:24529089–24563242) in the Nipponbare genome. (E) Predicted secondary structure of RNA encoded by chr12:22977717–22980753 of the Minghui 63 genome. (F) Expression values of the LIR in IMF2s of different genotypes quantified by mRNA sequencing. (G) Correlation coefficients between expression values of sRNAs regulated by Bin1556 and the LIR in 98 IMF2s. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

QTL analysis of variations in whole-genome sRNA species composition among different IMF2s. The 1,567 bins (QTL) are represented as vertical bars and arranged from left to right based on their genomic positions. The height of each bar indicates the LOD value of each bin while the width of each bar indicates the size of each bin. Adjacent chromosomes are denoted with different colors. The red rectangle in chromosome 9 indicates the bin harboring OsDCL2b while the red rectangle in chromosome 4 indicates the bin harboring OsRDR2.