Fig. 4: Identification of miR2118-dependent phasiRNAs in the anther wall.

a Size distribution of small RNAs from the WT (black line) and mi-1 (orange line) and mi-2 (yellow line) lines that were isolated from 0.5 mm anthers. b Pie charts summarizing the source of anther small RNAs from WT, mi-1, and mi-2. Anther small RNAs were mainly 21 nucleotides (nt) and derived from the lincRNAs/PHAS loci. c Venn diagram of overlapping anther 21-nt phasiRNA clusters and MEL1 21-nt phasiRNA clusters (germ-cell clusters). d, e Small RNA-seq reads of WT, mi-1, and mi-2 with two replicates that were mapped to the 21PHAS loci. Schematic alignment of 21-nt phasiRNAs is illustrated at the bottom by thick horizontal arrows with red (sense) and blue (antisense). Right panels depict the qPCR analysis of PHAS324 and PHAS1586  , lincRNAs, in 0.5 mm anthers at Stage 2. Bar represents mean ± SD (N = 3 or 4). Student’s t test. f Venn diagram of overlapping phasiRNA clusters that were identified in the anthers of WT and mi-1 and mi-2 lines. g Venn diagram shows overlapping miR2118-dependent clusters (648; f) and anther wall-specific phasiRNA clusters (532; c). The 320 overlapping clusters were considered miR2118-dependent phasiRNA clusters in the anther wall.