ARTICLE

https://doi.org/10.1038/s41467-021-25128-y OPEN Pre-meiotic 21-nucleotide reproductive phasiRNAs emerged in seed plants and diversified in flowering plants

Suresh Pokhrel 1,2, Kun Huang3, Sébastien Bélanger1, Junpeng Zhan 1,4, Jeffrey L. Caplan3, ✉ Elena M. Kramer5 & Blake C. Meyers 1,2

fi 1234567890():,; Plant small RNAs are important regulatory elements that ne-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots pro- duce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive pha- siRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

1 Donald Danforth Plant Science Center, Saint Louis, MO, USA. 2 Division of Plant Sciences, University of Missouri-Columbia, Columbia, MO, USA. 3 Bio- Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. 4 Department of Biology and Institute of Plant and Food Science, Southern University of Science and Technology, Shenzhen, Guangdong, China. 5 Department of Organismic and Evolutionary Biology, Harvard University, ✉ Cambridge, MA, USA. email: [email protected]

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hasiRNAs are generally produced by the action of 22 PHAS” loci) that were abundant specifically at the anther stage 7 nucleotide (nt) miRNA triggers on polyadenylated mes- (Fig. 1a). This stage corresponds to the pre-meiotic stage of P 10 senger RNA (mRNA) or long noncoding RNA targets anther development . These unannotated loci were mostly pre- generated by RNA Polymerase II1. These target transcripts are dicted to be noncoding (Supplementary Data 1); they contained subsequently processed by the action of RNA-DEPENDENT only one conserved sequence motif, which is the target site of RNA POLYMERASE 6 (RDR6) and DICER-LIKE (DCL) pro- miR11308-5p (henceforth, miR11308) (Fig. 1b, c). miR11308 has teins into duplexes of 21- or 24-nt small interfering RNAs (siR- three mature variants (Fig. 1b, d), two of which derive in the NAs). These siRNAs are “in phase” with one another; ï.e., the genome from polycistronic precursors (Supplementary Fig. 1A, B siRNAs map to the genome with regular spacing, the result of and Supplementary Data 2). Mature miR11308 accumulation processive cleavage by Dicer from a long precursor (a PHAS peaked at the anther stage 7 (Fig. 1b), similar to the peak of the precursor). The best characterized phasiRNAs in plants are trans- reproductive-enriched 21 nt phasiRNAs. For these loci, the most acting siRNAs, which function in development by regulation of abundant, phased 21 nt phasiRNA is generated from the cleavage auxin signaling1. site of miR11308 (or in a register spaced by 21 bp), as exemplified Reproductive tissues of grasses contain both 21 and 24 nt in Fig. 1e. These loci were distributed across all but chromosomes phasiRNAs, derived from noncoding precursors encoded at 2 and 3 of the F. vesca genome, whereas miR11308 originated hundreds of genomic loci. The 21 nt phasiRNAs are triggered by from chromosome 6 (Supplementary Fig. 1C). We were surprised miR2118 and are mainly enriched in pre-meiotic anther tissues, to find pre-meiotic 21 nt reproductive phasiRNAs, as we could during the stages at which the specification of cell fate occurs; find no record of a previous report of their presence in a eudicot. they originate in the epidermal layer of the anther but accumulate In maize and rice, mature variants of the miR2118 family subepidermally2. The 24 nt phasiRNAs are abundant in the trigger production of the 21 nt reproductive phasiRNAs2,4.In meiotic stages of anther tissues and are largely triggered by many other species, the miR482 family, a predecessor and relative miR2275, although in some monocots the miR2275 trigger is of the miR2118 family, triggers phasiRNAs from disease absent3. The spatiotemporal pattern of 21 nt phasiRNAs in male resistance genes11. However, in wild strawberry, only six reproductive tissues has been well-described in maize and rice2,4. miR2118/miR482-derived noncoding 21-PHAS loci are enriched These reproductive, 21 nt phasiRNAs lack obvious or, at least, during anther stages (Supplementary Data 1). We found 76 21- validated targets but play a role in photoperiod-sensitive male PHAS loci triggered by miR2118/miR482 in total; 65 loci are sterility in rice5. A mutant in rice of an Argonaute (AGO) protein abundant in vegetative tissues, whereas 11 loci are enriched in that selectively binds these 21 nt phasiRNAs6 is male sterile. reproductive tissues. These loci are mostly from protein-coding These data demonstrate that the 21 nt reproductive pre-meiotic genes and are mainly similar to genes encoding disease resistance phasiRNAs and/or their functions are required for male fertility, proteins, mirroring the 21-PHAS loci of other eudicots, such as and thus they are hypothesized to function in some important soybean and Medicago12,13 (Supplementary Fig. 2A and Supple- aspect of reproductive development. mentary Data 1). In strawberry, we found four precursors give The evolutionary origins of 21 nt reproductive phasiRNAs are rise to 15 mature variants of miR2118/miR482; these variants poorly examined. Their presence in monocots is clear, as the 21 nt accumulate in all tissue types, with greater abundances found in pre-meiotic phasiRNAs were first reported in grasses7 and later seeds (Supplementary Fig. 2B, C). Therefore, unlike grasses, in described from just three loci in asparagus but still enriched in the wild strawberry, the majority of 21 nt reproductive phasiRNAs middle layer, tapetum, and archesporial cells3. Despite extensive are triggered not by miR2118 but rather by a lineage-specific analyses, 21 nt reproductive phasiRNAs have not been reported in miRNA, miR11308. Furthermore, we validated the cleavage several well-studied plant families, including the Brassicaceae, directed by miR11308 and miR482/2118 family members of Fabaceae, and Solanaceae, leading to our own prior assumption these 21-PHAS loci using the degradome sequencing method that they are absent from the eudicots. In earlier work from our known as nanoPARE14 (Supplementary Fig. 3A, B and lab, we described a set of 21 noncoding loci in Norway spruce that Supplementary Data 3). are targets of miR2118, produce 21 nt phasiRNAs, and are enri- We next asked where this trigger of 21 nt reproductive ched in abundance in male cones8. This observation, as-yet phasiRNAs localizes. In situ hybridization localization of unsupported or reproduced from analyses of non-monocot miR11308 in wild strawberry showed that it localizes to microspore angiosperms, indicated the emergence of the 21 nt reproductive mother cells (MMCs), meiocytes, and tapetal cells (Fig. 2a, b) unlike phasiRNAs outside of the monocots, in gymnosperms. the miR2118 in maize, which localizes only in the epidermis2. Here we describe the discovery of the pathway of pre-meiotic, miR11308 is more abundant in MMCs of pre-meiotic cells and in 21 nt reproductive phasiRNAs in several eudicots as follows: wild tapetal cells of meiotic stages compared to the post-meiotic stage. strawberry (Fragaria vesca), rose (Rosa chinensis), the basal Next, we examined the localization patterns of the most abundant eudicot columbine (Aquilegia coerulea), and flax (Linum usita- 21 nt phasiRNAs from these loci with single molecule fluorescence tissimum). We conclude that similar to the 24 nt reproductive in situ hybridization (smFISH) (Supplementary Fig. 4A) using a phasiRNAs9, the 21 nt reproductive pathway is widespread in pool of fluorescently labeled probes against 50 21 nt phasiRNAs. angiosperms and may even, with origins in seed plants, have Surprisingly, we found these molecules were localized in all cell emerged prior to the 24 nt reproductive phasiRNAs. layers, most abundant in the MMC during the pre-meiotic stage of anther development (Fig. 3a). By the tetrad stage, the phasiRNAs are only detectable in the MMCs and barely detectable in other cell Results layers. This spatiotemporal distribution of 21 nt phasiRNAs is Pre-meiotic anthers of wild strawberry produce abundant 21 nt generally similar to the accumulation pattern found in grasses2,15 phasiRNAs. We analyzed small RNAs (sRNAs) in tissues of wild and thus these 21 nt phasiRNAs in strawberry may play a role in (diploid) strawberry (F. vesca), as this plant was highly infor- male fertility. mative for our earlier analyses of 24 nt reproductive phasiRNAs9; in fact, we were considering it as a possible model system for the study of 24 nt phasiRNAs. We characterized 21 nt phasiRNAs in Conservation of miR11308 and 21 nt reproductive phasiRNAs both vegetative and reproductive tissues of wild strawberry, and in other Rosaceae species. To determine whether this pathway is we identified 25 loci that give rise to 21 nt phasiRNAs (“21- conserved in other members of the Rosaceae family and beyond,

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a d 21PHAS_12 14 21PHAS_73 21PHAS_63 12 21PHAS_71 miR11308a 21PHAS_69 10 miR11308b 21PHAS_67 miR11308c 21PHAS_11 8 21PHAS_76 Consensus 21PHAS_13 6 21PHAS_72 21PHAS_50 4 21PHAS_61 21PHAS_66 2 21PHAS_74 e 21PHAS_90 21PHAS_61; 6:3446776-3447323 0 21PHAS_64 21PHAS_62 21PHAS_68 <=20 21 22 23 24 >=25 21PHAS_75 150+ 21PHAS_41 21PHAS_40 100 21PHAS_65 21PHAS_101 50 21PHAS_9 21PHAS_45 0

ower 0 Leaf e-meiotic Seedling wall_10D 50 ReceptacleSeeds_4DSeeds_10Dry_wall_4D va b vary_ Athst8_meioticAthst9_meiotic O O Unopened_FlAthst6_prAthst7_pre-meiotic 100 5 Labels 150+ 4 miR11308a

3 miR11308b f

2 miR11308c 37.5+

log10(RP20M) 25 1 12.5

0 0 0 phasing score abundance sRNA Leaf 12.5 all_4D -meiotic Seedling w e ReceptacleSeeds_4DSeeds_10Dy_ ar 25 v Athst8_meioticAthst9_meiotic O vary_wall_10D O c Unopened_FlowerAthst6_prAthst7_pre-meiotic 37.5+

Fig. 1 Reproductive 21 nt phasiRNAs triggered by miRNA miR11308 are abundant in wild strawberry. a Expression of 21 nt reproductive phasiRNAs in different tissues and anther development stages. The key at the right indicates the abundance in units of log2(RP20M). RP20M: reads per 20 million mapped reads. Athst: anther stage. b Abundance of miR11308 members in log10(RP20M) in different tissues of wild strawberry. c Above: sequence logo denoting conservation of target site of miR11308 for 25 21-PHAS loci. Below: nucleotide sequence alignment of 21-PHAS loci with sequence similarity denoted by the intensity of the blue color showing that the miR11308 target site is the only conserved region for all the precursors. d Alignment of members of the miR11308 family in wild strawberry. The degree of conservation is denoted by intensity of the blue color; the consensus sequence of the alignment is shown with a sequence logo. e Abundance (RP15M) of small RNAs in both strands of an example locus padded with 500 base pairs, each side. sRNA sizes are denoted by colors, as indicated at the top. f Phasing score of same locus as e; the red dot indicates the highest phased sRNA position. The red dot also represents the coordinate (3,446,776), which exactly coincides with the predicted cleavage site of miR11308, denoted by the red arrow. Source data underlying Fig. 1a, b are provided as a Source Data file.

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a E En M sRNA-FISH T

MMC

b miR11308 Tapetal cell (zoom in) Negative control

Pre-meiotic stage

Meiotic stage

Post-meiotic stage

10 µm 5 µm 10 µm

Fig. 2 miR11308 accumulates in microspore mother cells, meiocytes, and tapetal cells in the anthers of wild strawberry. a Schematic diagram of the sRNA-FISH method in anther stages shown in b. E: epidermis, En: endodermis, M: middle layer, T: tapetum, MMC: microspore mother cell. b In situ hybridization of miR11308 in pre-meiotic, meiotic, and post-meiotic anther stages. The experiment was repeated twice with similar results. we investigated the presence of the miR11308 in species of We next analyzed the sRNA transcriptomes of rose and Rosaceae and Betulaceae for which a genome sequence is avail- identified nine 21-PHAS loci enriched in the anthers of early able. We found that the pattern of miR11308 presence is con- reproductive bud stages (Fig. 3c and Supplementary Fig. 4B), thus sistent with its emergence in the Rosaceae subfamily Rosoideae, as confirming the presence of 21 nt reproductive phasiRNAs in this it is present in at least four genera (Fig. 3b and Supplementary species. Similar to wild strawberry, these loci are mostly Data 2). Similar to miR22759, this miRNA generates mature noncoding (Supplementary Data 4) and are also targeted by sequences with a polycistronic precursor cluster, which itself is two variants of miR11308 (Fig. 3d). We further found that three conserved in the Rosoideae. We hypothesize that this poly- precursors of six mature variants of the rose miR2118/482 family cistronic nature of the precursors is of functional importance for target 86 21-PHAS loci; we found them to be abundant in all the biogenesis of reproductive phasiRNAs. tissue types and mostly derive from genes coding for nucleotide-

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a 50-phasiRNAs MMC / zoom in Negative control

Pre-meiotic stage signal

signal Meiotic/tetrad stage

10 µm 5 µm 10 µm

b c

11308 miR MIR Cluster Betula nana No No Magnoliopsida Pyrus communis No No Prunus persica No Amygdaloideae No 3 Malus domestica No No Rubus occidentalis Yes Yes Rosaceae Rosa chinensis Rosoideae Yes Yes Potentilla micrantha Yes Yes 2 Fragaria vesca Yes Yes Fragaria ananassa Yes Yes log10(RP20M) 1

d rch-miR11308a 0 rch-miR11308b

Leaf Root Petal Pollen Stamen consensus Bst_1_anthBst_2_anthBst_3_anthBst_4_anth

Fig. 3 The 21 nt reproductive phasiRNAs localize in all cell layers of the pre-meiotic strawberry anthers and the miR11308 trigger emerged in the Rosoideae. a Localization of abundant 50 21 nt reproductive phasiRNAs in pre-meiotic and meiotic anther tissues of wild strawberry. The localization experiment was repeated twice with similar results. b Phylogenetic tree showing conservation of miR11308 in the Rosaceae. c Abundances of summed 21 nt phasiRNAs in different tissues of rose in units of log10(RP20M). In the boxplot, the center line represents the median, box limits are the upper and lower quartiles; whiskers are the 1.5× interquartile range of upper or lower quartiles; points show the scatter of data points for nine (n = 9) 21-PHAS loci. Bst indicates bud stage, anth indicates anther. d Alignment of members of miR11308 in rose. Source data underlying Fig. 3c are provided as a Source Data file. binding leucine-rich repeat proteins otherwise known as “NLRs,” nt reproductive phasiRNAs are present and/or conserved in this which commonly function as innate immune receptors (Supple- species. We identified 112 21-PHAS loci; 91 enriched in repro- mentary Fig. 4C, D and Supplementary Data 4). Hence, we report ductive tissues (Fig. 4a) and an additional 21 21-PHAS loci that that the miR11308 has acquired a role to generate 21 nt were expressed similarly in all tissue types (Supplementary reproductive phasiRNAs in the Rosaceae subfamily Rosoideae. Fig. 5B). Out of 112 loci, we found that 65 loci are targeted by miR14051; most loci (63/65) are from noncoding regions of the Trigger miRNAs and conservation of 21 nt reproductive pha- genome (Supplementary Data 5) and they possess a conserved 22 siRNAs in the basal eudicot columbine and in flax. We next nt motif: the target site of miR14051 (Supplementary Fig. 5C). examined the sRNA transcriptomes of vegetative tissues and four The trigger-target cleavage was detected in 33 (of 65) 21-PHAS different bud stages (Supplementary Fig. 5A) of the basal eudicot loci using nanoPARE sequencing (Supplementary Data 6). The Aquilegia (common name “columbine”) to determine whether 21 miR14051 miRNA has six mature variants from seven different

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a b 21PHAS_4 14 21PHAS_32 aco-miR14051a 21PHAS_83 21PHAS_24 aco-miR14051b 21PHAS_16 12 aco-miR14051c,g 21PHAS_88 aco-miR14051d 21PHAS_70 10 21PHAS_77 aco-miR14051e 21PHAS_26 aco-miR14051f 21PHAS_27 8 21PHAS_65 21PHAS_66 consensus 21PHAS_64 6 21PHAS_72 21PHAS_38 21PHAS_97 4 21PHAS_31 21PHAS_15 20 21PHAS_40 2 21PHAS_21 21PHAS_86 21PHAS_29 0 21PHAS_2 21PHAS_87 15 21PHAS_19 21PHAS_51 Labels 21PHAS_30 aco-miR14051a 21PHAS_6 aco-miR14051b 21PHAS_43 21PHAS_14 10 aco-miR14051c,g 21PHAS_82 aco-miR14051d 21PHAS_59 aco-miR14051e 21PHAS_23 log10(RP20M) 21PHAS_57 aco-miR14051f 21PHAS_46 21PHAS_33 5 21PHAS_36 21PHAS_25 21PHAS_5 21PHAS_42 21PHAS_81 21PHAS_7 0 21PHAS_84 21PHAS_50 21PHAS_41 21PHAS_85 Leaf Root Bst_1 21PHAS_79 Bst_2 21PHAS_60 Bst_3_anthBst_4_anth 21PHAS_74 21PHAS_71 21PHAS_8 c 21PHAS_17 aco_miR2118a 21PHAS_3 21PHAS_35 aco_miR2118b,c,f,i 21PHAS_49 aco_miR2118d 21PHAS_58 aco_miR2118e 21PHAS_18 21PHAS_69 aco_miR2118g 21PHAS_34 aco_miR2118h 21PHAS_45 21PHAS_107 consensus 21PHAS_39 21PHAS_48 21PHAS_76 21PHAS_109 21PHAS_110 15 21PHAS_73 21PHAS_78 21PHAS_68 21PHAS_111 21PHAS_1 21PHAS_28 21PHAS_89 21PHAS_108 10 21PHAS_55 Labels 21PHAS_37 aco_miR2118a 21PHAS_61 21PHAS_91 aco_miR2118b,c,f,i 21PHAS_56 aco_miR2118d 21PHAS_105 aco_miR2118e 21PHAS_90 21PHAS_95 log10(RP20M) aco_miR2118g 21PHAS_101 5 aco_miR2118h 21PHAS_9 21PHAS_112 21PHAS_94 21PHAS_54 21PHAS_96 21PHAS_102 21PHAS_53 21PHAS_67 0

Leaf Root Bst_1 Bst_2 Leaf Root Bst_1 Bst_2 Bst_3_anthBst_4_anth Bst_3_anthBst_4_anth pre-meiotic meiotic post-meioitic

Fig. 4 Conservation of reproductive 21 nt phasiRNAs and their triggers in the basal eudicot columbine. a Expression of 21 nt reproductive phasiRNAs in different vegetative tissues and anther development stages in columbine. The lowermost ten loci are most enriched in stage 4. The yellow gradient arrow indicates the developmental stages of anthers. The key at the right indicates the abundance in units of log2(RP20M). Bst indicates bud stage, anth indicates anther. b Above: alignment of members of miR14051 in columbine. The degree of conservation is denoted by the intensity of the blue color and the consensus sequence of the alignment is shown in a sequence logo. Below: abundance of miR14051 members in log10(RP20M) in different tissues of columbine. c Similar to b but for the miR2118/482 family in columbine. Source data are provided as a Source Data file.

6 NATURE COMMUNICATIONS | (2021) 12:4941 | https://doi.org/10.1038/s41467-021-25128-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25128-y ARTICLE precursors (Fig. 4b and Supplementary Data 2), with one poly- phasiRNAs are produced by DCL4, which is conserved in seed cistronic cluster generating two mature variants (Supplementary plants (Supplementary Fig. 10B) and mostly enriched in repro- Fig. 5D). The accumulation pattern of miR14051 is similar to the ductive tissues, except in rose (Supplementary Data 8). DCL3 and 21 nt phasiRNAs initiated by this miRNA (Fig. 4a, b). DCL5 are Dicer proteins responsible for the production of 24 nt The remaining 47 (of 112) 21-PHAS loci are targeted by siRNAs, with DCL5 specialized for 24 nt phasiRNAs in monocots mature variants of the miR2118/482 family (Fig. 4a, c and and DCL3 hypothesized to functioning in dual roles, producing Supplementary Fig. 6A). Among these, 37 loci are abundant in 24 nt phasiRNAs in eudicots in addition to its well-described role bud stage 2 and 3 (Fig. 4a), and are noncoding, whereas the other in making heterochromatic siRNAs2,9. There is a duplication of 10 loci are abundant in all tissue type and are mostly derived from DCL3 in all four eudicot genomes that we examined for pha- NLR protein-coding genes (Supplementary Fig. 5A and Supple- siRNAs and in a gymnosperm, ginko, among all the species; mentary Data 5). NanoPARE sequencing was used to validate the duplicated DCL3 (DCL3b) was more enriched in reproductive trigger-target interaction of 25 (of 47) 21-PHAS loci (Supple- tissues than DCL3a (Supplementary Data 8), suggesting the mentary Data 6). The miR2118/482 family has six mature possibility of neo-functionalization of DCL3b for the production variants that are derived from nine different precursors with two of 24 nt reproductive phasiRNAs in eudicots9. polycistronic clusters (Fig. 4c, Supplementary Fig. 6B, and We identified 12, 13, and 17 AGO proteins encoded in the wild Supplementary Data 2). The miR2118/482 family members are strawberry, rose, columbine, and flax genomes, respectively highly abundant at bud stages 2 and 3, similar to the phasiRNAs (Fig. 5a). All of the identified AGO transcripts are expressed they trigger (Fig. 4c). Most of the reproductive 21-PHAS loci in ≥0.5 TPM (transcripts per million) in these four species (Fig. 5b). Aquilegia are concentrated on chromosome 4, an unusual In rice, AGO1b/1d were implicated in loading miR2118 and chromosome showing higher polymorphism compared to other U-21-nt phasiRNAs15, whereas AGO5c (i.e., rice MEL1) is chromosomes between the ten different Aquilegia species16. The implicated for the loading of reproductive C-21-nt phasiRNAs6,15. miR14051 miRNA precursors were derived from chromosomes 4 Here we identified two AGO5 copies in strawberry and both and 1, whereas miR2118/482 precursors were distributed on AGO5 copies are enriched in pre-meiotic stages, whereas in rose, chromosomes 1, 3, and 5 (Supplementary Fig. 7). Unlike grasses, among three AGO5 copies, only AGO5b (orthologous to AGO5b in Aquilegia, 21 nt reproductive phasiRNAs are triggered by two in strawberry) was enriched in reproductive tissues. In flax, one miRNA family members, for which at least one precursor is copy of AGO5 is enriched in the reproductive tissues. In polycistronic. columbine, both copies of AGO5 are enriched in the reproductive To more precisely identify the developmental stages of the tissues but AGO5b was more enriched (Supplementary Data 8). buds/anthers that we sequenced, anthers from seven bud lengths Overall, based on phylogeny and their expression profiles, AGO5 were examined. We found anthers from 2 to 4 mm buds are in or AGO5a/b are the candidate AGO proteins for loading pre- pre-meiotic stages, whereas 5–6 and 10–20 mm buds are meiotic meiotic 21 nt phasiRNAs in eudicots as they are in monocots. and post-meiotic stages, respectively (Supplementary Fig. 8). Among AGO1 family members, in columbine, AGO1c is more Therefore, the 21 nt phasiRNAs in columbine initiate in pre- enriched in reproductive stages, whereas in flax, strawberry, and meiotic stages and their accumulation is maintained up to post- rose, AGO1a is more enriched in the reproductive tissues (Fig. 5b meiotic stages. and Supplementary Data 8). Thus, AGO1c for columbine, and To further confirm the conservation of the 21 nt reproductive AGO1a for other three eudicots might be the effector protein for phasiRNA pathway in eudicots, we profiled sRNAs from both triggers of 21 nt reproductive phasiRNAs. Orthologs of AGO18 vegetative and reproductive tissues of flax, a species in the are absent in eudicots, gymnosperms, Amborella, and Asparagus phylogenetic tree of eudicots outside the Rosaceae but within the consistent with its hypothesized origin in grasses (Fig. 5a). Genes rosids. We found 11 reproductive-enriched 21-PHAS noncoding encoding members of the AGO6 clade (specifically AGO6a) were loci triggered by miR2118/482 variants (Supplementary enriched more than AGO4 in the reproductive tissues, whereas Fig. 9A–C) in flax (Supplementary Data 2 and 7). Unlike AGO9 in rose and strawberry are expressed exclusively in the columbine, in flax, these 21-PHAS loci are triggered only by the reproductive tissues (Fig. 5b and Supplementary Data 8). It is canonical trigger of pre-meiotic phasiRNAs, as described from known that AGO4, AGO6, or AGO9 are binding molecules for 24 extensive work in monocots2,4 (miR2118/miR482). nt heterochromatic siRNAs during RNA-directed DNA methylation17. Thus, based on their gene expression and enrichment, AGO9 and AGO6 may also act as the effector Genes important to the 21 nt reproductive phasiRNA pathway. molecule for reproductive 24 nt phasiRNAs in eudicots. Factors known to function in phasiRNA biogenesis are mainly RDR6, AGOs, and DCL proteins. We analyzed the presence of these proteins in a total of 13 species: 4 eudicots (wild strawberry, Discussion rose, columbine, flax) we analyzed for phasiRNAs, 2 gymnos- We found that 21 nt reproductive phasiRNAs are present in perms (Norway spruce, ginko), and another 7 representative several eudicots: wild strawberry, rose, the basal eudicot colum- species for which these genes were already characterized— bine, and flax. Whereas previous studies have shown that 21 nt Amborella, soybean, tomato, Arabidopsis, Asparagus, maize, and reproductive phasiRNAs exist in one gymnosperm8 and more rice (Fig. 5a and Supplementary Fig. 10A, B). RDR6 is responsible widely in monocots2,4,7,18,19 (Fig. 6a), this pathway has not been for making the double-stranded RNA precursors after miRNA described in several well-studied eudicot families including the cleavage during phasiRNA biogenesis and it is conserved in seed Brassicaceae, Fabaceae, and Solanaceae2,12,13. Another class of 21 plants (Supplementary Fig. 10A). Among 13 species, RDR6 is or 22 nt secondary siRNAs active in plant reproduction are the present in two or more copies in all species, except in rice, epigenetically activated siRNAs (easiRNAs) that accumulate in Asparagus, Arabidopsis, and columbine. The three eudicot species Arabidopsis during stages of pollen maturation, derived from we analyzed for phasiRNAs have two RDR6 copies, but not activated transposable elements20,21. easiRNAs appear to play a columbine (Supplementary Fig. 10A). We hypothesize that there role in male gamete production and possibly post-fertilization is a species-specific duplication of RDR6, potentially facilitating a genome stability and seed viability22. Even though both pha- sub-functionalization of their roles, as both copies are enriched in siRNAs and easiRNAs are secondary siRNAs important for plant reproductive tissues (Supplementary Data 8). The 21 nt reproduction, there is no evidence suggesting that they are related

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a

AcAGO6f SlAGO15

A SlAGO4b SlAGO4c LuAGO4b SlAGO4a GmAGO4b LuAGO4a GmAGO4a o

A GmAGO4c AtAGO4

G

ZmAGO4aFvAGO4 O RcAGO4

6 AGO8/9 OsAGO4aZmAGOb

ZmAGO6 AcAGO6d AcAGO6b ZmAGO4c AcAGO6c AcAGO6e AcAGO6a SlAGO6 OsAGO4b GmAGO6a AtAGO6 AoAGO4a LuAGO6a LuAGO6c AoAGO4b LuAGO6b FvAGO6c AcAGO4a RcAGO6c RcAGO6b AcAGO4b RcAGO6a FvAGO6a FvAGO6b AtAGO9 AtrAGO6 AtAGO8 PaAGO6b AGO4 RcAGO9 PaAGO6a GbAGO6 FvAGO9 GbAGO7 FvAGO8 PaAGO7 RcAGO8 AtrAGO7 GmAGO9 AoAGO7 ZmAGO7 AGO15/6 SlAGO9 OsAGO7 AcAGO7 AtAGO7 GmAGO10e LuAGO7c LuAGO7a GmAGO10f LuAGO7b GmAGO10h RcAGO7 GmAGO10g SlAGO7 AGO6 SlAGO10b GmAGO7b GmAGO7a AcAGO10b GbAGO2c ZmAGO10a GbAGO2b ZmAGO10b GbAGO2a OsAGO10 PaAGO2a AGO7 PaAGO2c AoAGO10 PaAGO2d AtrAGO10b PaAGO2b GmAGO10d PaAGO2e GmAGO10c AtrAGO2 AoAGO2 AGO2/3 GmAGO10a ZmAGO2b GmAGO10b ZmAGO2a FvAGO10 OsAGO3 LuAGO10b OsAGO2 LuAGO10a AcAGO2 AtAGO10 SlAGO2a SlAGO3 AGOx SlAGO10a SlAGO2b AcAGO10a GmAGO2 AtrAGO10a GmAGO3 PaAGO10 RcAGO2 GbAGO1 FvAGO2 AtAGO3 AGO18 PaAGO1 AtAGO2 AtrAGO1 LuAGO2b AcAGO1c LuAGO2a AtrAGOx AcAGO1b OsAGO18 AcAGO1a ZmAGO18b LuAGO1b ZmAGO18c ZmAGO18a AGO5 LuAGO1c AtrAGO5a LuAGO1d AtrAGO5b AoAGO5c LuAGO1a AoAGO5a AoAGO5b GmAGO1b ZmAGO5a OsAGO5a GmAGO1aSlAGO1b ZmAGO5d AGO1 OsAGO5e SlAGO1a OsAGO5b ZmAGO5b ZmAGO5c RcAGO1a OsAGO5c AtAGO1 OsAGO5d FvAGO1a LuAGO5 AcAGO5b AcAGO5a SlAGO5 AtAGO5 GmAGO5a

R GmAGO5b FvAGO1b FvAGO5b

RcAGO1b c

ZmAGO1b A

ZmAGO1c G AGO10 OsAGO1b

ZmAGO1a O OsAGO1a OsAGO17

AoAGO1a 5 AoAGO1b AoAGO1c

b OsAGO1d

OsAGO1c RcAGO5c ZmAGO1d FvAGO5a RcAGO5a

bdce

96.0 108.1 42.4 35.8 23.6 AcAGO1a 19.2 18.8 27.6 7.6 LuAGO1a 40.5 283.2 126.2 110.1 75.2 FvAGO1a 131.3 68.2 50.6 50.7 RcAGO1a 162.4 173.1 90.2 91.8 24.8 AcAGO1b 25.5 21.6 21.5 13.5 LuAGO1b 27.9 109.3 59.7 47.4 59.7 FvAGO1b 12.7 12.5 7.2 23.6 RcAGO1b 26.7 43.7 2.0 3.0 0.0 AcAGO1c 26.0 19.5 20.8 11.5 LuAGO1c

14.5 15.5 13.3 14.9 RcAGO2 21.8 44.5 43.8 31.4 21.7 FvAGO2 2.0 1.8 0.6 0.4 0.7 AcAGO2 13.1 13.6 15.5 9.2 LuAGO1d

86.4 43.3 35.3 37.9 RcAGO4 54.3 41.9 14.5 8.2 6.8 AcAGO4a 4.6 0.0 0.0 0.0 LuAGO2a 90.2 299.3 167.0 149.0 127.1 FvAGO4 96.8 87.2 44.8 12.8 15.2 AcAGO4b 1.2 0.0 0.0 0.0 LuAGO2b 3.2 1.6 1.5 6.3 RcAGO5a 2.3 0.2 0.6 0.8 0.1 FvAGO5a 74.0 69.7 15.5 116.4 10.7 AcAGO5a 33.5 26.5 31.2 12.8 LuAGO4a 68.6 26.7 17.5 14.1 RcAGO5b 24.0 20.2 5.7 11.4 0.1 AcAGO5b 45.2 34.0 37.5 14.9 LuAGO4b 15.0 46.5 34.4 31.0 21.0 FvAGO5b 4.7 2.1 1.8 5.8 RcAGO5c 8.1 13.3 4.7 2.7 1.5 AcAGO6a 6.3 10.0 15.3 0.4 LuAGO5 17.2 56.1 17.5 11.1 1.2 FvAGO6a 26.3 30.7 13.0 4.8 14.2 AcAGO6b 0.5 0.4 0.4 0.0 LuAGO6a 11.5 6.0 2.7 0.2 RcAGO6a 18.5 36.1 14.9 17.3 15.1 FvAGO6b 52.0 102.8 35.1 15.3 5.0 AcAGO6c 0.8 0.7 0.7 0.2 LuAGO6b 2.1 2.8 1.6 0.2 RcAGO6b 10.6 14.1 6.6 2.2 1.5 AcAGO6d 0.7 0.4 0.4 0.2 LuAGO6c 14.3 80.7 14.9 7.6 8.1 FvAGO6c 9.2 4.7 3.8 1.3 RcAGO6c 5.8 9.8 4.6 1.8 2.0 AcAGO6e 2.3 1.8 1.9 0.9 LuAGO7a

1.4 0.6 5.4 20.9 0.2 FvAGO8 0.9 0.9 0.4 1.1 0.0 AcAGO6f 2.7 2.1 2.3 1.1 LuAGO7b 5.7 3.5 1.8 1.6 RcAGO7 12.5 12.5 3.8 2.1 3.1 AcAGO7 2.4 2.0 3.0 4.0 LuAGO7c 5.6 0.0 26.9 0.7 0.0 FvAGO9 1.4 4.3 2.2 0.1 RcAGO8 51.9 52.9 20.1 2.9 1.0 AcAGO10a 10.6 8.1 9.4 3.3 LuAGO10a

74.9 77.6 30.1 35.2 74.3 FvAGO10 1.6 0.2 0.5 0.0 RcAGO9 8.8 8.5 2.3 0.7 0.1 AcAGO10b 13.9 9.5 10.7 3.6 LuAGO10b

Leaf Leaf Leaf Bst_1 Bst_2 Leaf Bst_1 Bst_2Bst_3 Anther_9 Anther6_7Anther7_8 Anther_10 Bst_1_anthBst_2_anthBst_3_anth Bst_3_anthBst_4_anth

Fig. 5 Characterization of AGO gene family members in wild strawberry, rose, columbine, flax, and other representative species from gymnosperms, eudicots, and monocots. a Phylogenetic tree of Argonaute protein family members encoded in the genomes of plant species, with two-letter prefix indicating the source genome: wild strawberry (Fv), rose (Rc), columbine (Ac), flax (Lu), Norway spruce (Pa), and ginko (Gb) identified in this study along with other seven representative species—Amborella (Atr), soybean (Gm), tomato (Sl), Arabidopsis (At), asparagus (Ao), maize (Zm), and rice (Os). Abundance profile of genes encoding AGO family members in b wild strawberry, c in rose, d in columbine, and e in flax. Bst indicates the bud stage, anth indicates the anther. Source data underlying Fig. 5b–e are provided as a Source Data file.

8 NATURE COMMUNICATIONS | (2021) 12:4941 | https://doi.org/10.1038/s41467-021-25128-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25128-y ARTICLE

classes of RNAs, nor is it evident that easiRNAs are found outside Moreover, the miR2118/482 family triggers 21 nt phasiRNAs of Arabidopsis. We hypothesize that 21 nt reproductive pha- from NLR genes in columbine, a conserved role of this miRNA siRNAs are a more fundamental and broadly conserved pathway, from gymnosperms to angiosperms, particularly in eudicots1. The which emerged in gymnosperms but were lost in some lineages miR2118/482-derived NLR phasiRNA pathway is also found in due to accommodations or adaptations of development. wild strawberry and rose; although the role of this miRNA family In the basal eudicot columbine, we showed that reproductive in generating reproductive 21 nt phasiRNAs is almost completely tissues produce abundant phasiRNAs generated by both the shifted to miR11308, a non-canonical trigger of reproductive canonical trigger miR2118/482 and a lineage-specific miRNA, phasiRNAs, we also found evidence for the canonical trigger of miR14051, indicating a division of this phasiRNA biogenesis role. reproductive phasiRNAs in wild strawberry miR2118. In flax, we

NATURE COMMUNICATIONS | (2021) 12:4941 | https://doi.org/10.1038/s41467-021-25128-y | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25128-y

Fig. 6 Conservation of reproductive 21 nt phasiRNA in seed plants. a Phylogeny showing the representative species of seed plants according to estimated divergence times based on the time tree of life (Kumar et al.51). The miR2118/482 superfamily consists of miR2118, miR482, miR8558, and miR472. MYA means million years ago. √ indicates the presence and X indicates the absence; na indicates “not analyzed”. b Model of generation of 21 nt reproductive phasiRNAs in seed and flowering plants. miRNA triggers initiate the cleavage of Pol II transcripts with the help of AGO proteins resulting 3′-end cleaved fragments, which are processed by RDR6 to generate double-stranded RNA molecules. DCL4 chops these double-stranded molecules into 21 nt duplexes, initiated from the cleavage site (denoted by dotted lines). In gymnosperms, monocots, and some eudicots such as flax, miR2118 is the only trigger for the production of 21 nt reproductive phasiRNAs, whereas in columbine, rose, and wild strawberry, there is a lineage-specific miRNA trigger for this role. Based on the roles in other species, family members of AGO1 and AGO5 might load the trigger miRNA and 21 nt phasiRNAs, respectively. The resulting phasiRNAs from these mechanisms were predicted to act in both cis and trans to modulate the gene expression based on the studies in monocots. Panel b was created with BioRender.com under an academic license.

found that only miR2118/482 triggers the production of 21 nt using a Qubit Fluorometer (ThermoFisher Scientific, catalog number #Q33238). reproductive phasiRNAs, consistent with a conserved role of the Flax sRNAs (20–30 nt) were size-selected in a 15% polyacrylamide/urea gel and canonical trigger in this species. Based on these data, we propose used for sRNA library preparation, whereas for other species direct total RNA was used for sRNA library construction. An aliquot of 5 µg of total RNA was used for a model of how different triggers in different species function to size selection. generate reproductive 21 nt phasiRNAs in seed and flowering plants (Fig. 6b). All of these triggers starts with uracil (U) and are RNA libraries and sequencing. Flax sRNA libraries were constructed using the 22 nt in length consistent with earlier finding that only the 22 nt NEBNext Small RNA Library Preparation set for Illumina (NEB, catalog number form of miRNAs in AGO1 complex are capable of initiating #E7300); for columbine and rose, RealSeq-AC miRNA Library kit for Illumina phasiRNA biogenesis23. Based on the roles in other species6,24,25, (Somagenics, catalog number #500-00012) was used as per the manufacturer’s recommendation. RNA-sequencing (RNA-seq) libraries were constructed using family members of the AGO5 clade might load 21 nt phasiRNAs NEBNext Ultra II Directional RNA Prep kit for Illumina; RNA was treated with that were predicted to act in both cis and trans, to modulate DNase I (NEB, catalog number #M0303S) and then cleaned using the RNA Clean transcript levels. Clearly, more extensive data, from more diverse & Concentrator™-5 (Zymo Research, catalog number #R1015S). sRNA- and RNA- genomes of seed plants, would help improve the granularity of seq libraries were single-end sequenced with 76 cycles. All libraries were sequenced on an Illumina Nextseq 500 instrument at the University of Delaware Sequencing this model. and Genotyping Center at the Delaware Biotechnology Institute. For strawberry Similar to the 21 nt reproductive phasiRNAs in grasses, in wild vegetative/reproductive tissues9,26,27 and rose vegetative tissues28, sRNA- and strawberry, these phasiRNAs are also localized in all cell layers RNA-seq data were downloaded from public databases and used for the analysis. and are abundant in MMCs of pre-meiotic anther, suggesting similar role in reproductive development of eudicot anthers— sRNA data analysis and PHAS annotation. Raw reads of sRNA libraries were whatever that role may be. Even more than a decade after processed by an in-house preprocessing pipeline29. PHASIS30 with a p-value cutoff 31 describing the 21 nt reproductive phasiRNAs in rice and maize7, of 0.001 and ShortStack with default parameters were used to identify the PHAS loci in all of the species. PHAS loci from two different software were merged using their precise molecular activities are unclear. Unlike miR2118 in BEDTools32.OnlyPHAS loci having target sites for miRNA triggers were anno- grasses, which localizes to the epidermis, miR11308 localizes in tated as valid PHAS loci. Target prediction was carried out by sPARTA33 with a MMCs in pre-meiotic anthers, suggesting MMCs as the origin of target cutoff score of ≤4. The sRNA abundance and phasing score were viewed at 34 these pre-meiotic phasiRNAs. The localization of miR11308 in customized browser . Based on the target site of triggers, 50 bp upstream and 500 bp downstream strand-specific sequences were extracted from each PHAS loci and tapetal cells of meiotic anthers suggests other potential roles of were annotated de novo by BLASTX35 against UniRef90. The coding potential of this miRNA in later stages of anther development, yet to be PHAS loci were assessed for protein-coding potential by CPC (Coding Potential examined. Calculator)36. In conclusion, the emergence and conservation of 21 nt reproductive phasiRNAs in eudicots supports an origin in the RNA-seq data analysis and visualization. Raw reads were processed by an in- seed plants and suggests a functional role in reproduction. In rice, house preprocessing pipeline and Hisat237 was used to align the reads with respective genomes using default parameters. Raw counts were obtained by using mutations in these 21-PHAS loci cause photoperiod-sensitive TPMCalculator38 and normalized to TPM. To visualize the conservation of the 5 male sterility , a key feature that has been leveraged for hybrid target site of miRNA triggers, a multiple sequence alignment of PHAS loci strand- seed production. Therefore, the study of these 21-PHAS loci in a specific sequences was performed using MUSCLE39 with default parameters and broader set of plant species may open up avenues to improve we visualized the alignment using Jalview40. Weblogo images were created using plant yields. Currently, it is unknown how these phasiRNAs logo generation form (http://weblogo.berkeley.edu/logo.cgi). Circular plots were made using OmicCircos41 for the chromosomal distributions, and pheatmap modulate male fertility; future studies are needed to produce a (https://rdrr.io/cran/pheatmap/) and ggpubr (https://github.com/kassambara/ deeper understanding of reproductive phasiRNAs. Some species ggpubr) were used to draw heatmaps and boxplot in R. may be better models than others, due to their life history, miRNA triggers, PHAS locus complexity, or other traits, as Phylogenetic analysis. Identification of RDR, Dicer, and AGO families for all of demonstrated by the diversity we observed in eudicots. the species were carried out by using Orthofinder42, except for Norway spruce (Pica abies) for which genBlastG43 was used. The resulting protein sequences were visualized in CDvist44 to verify complete domains. Curated proteins were aligned 45 Methods using default settings in PASTA . The maximum likelihood gene tree for each protein family were generated using RAxML46 over 100 rapid bootstraps with Plant material harvesting and RNA isolation. Vegetative tissues and unopened options “-x 12345 -f a -p 13423 -m PROTGAMMAAUTO.” Trees were visualized flower buds/anthers of columbine (cultivar, “origami”) were collected from and manipulated in iTOL47. The annotations used for the phylogenetic analysis for greenhouse-grown plants under conditions of 20/18 °C day/night and 16/8 h light/ all 13 species for AGO, DCL, and RDR proteins are listed in Supplementary dark. A total of four different bud stages (Supplementary Fig. 5A) were char- Data 9–11, respectively. acterized and buds were harvested for stage 1 and 2, and from stage 3 and 4, anthers were harvested. Two biological replicates were sequenced for each stage. Flax was grown in a growth chamber with 12 h light at 25 °C followed by 12 h dark at 20 °C. Tissue embedding and microscopy. Fresh anthers were dissected from different Three bud stages (Supplementary Fig. 8A) were harvested and frozen immediately bud stages and fixed in a FAA (Formaldehyde Alcohol Acetic Acid, 10%:50%:5% + in liquid nitrogen. Rose anthers from four different bud stages (Supplementary 35% water) solution overnight and dehydrated through a standard acetone series Fig. 4B) were harvested and immediately frozen in liquid Nitrogen. Total RNA was (30%, 50%, 70%, 80%, 90%, and 100% of cold acetone) prior to being resin infil- isolated using the PureLink Plant RNA Reagent (ThermoFisher Scientific, catalog trated and embedded using the Quetol (Electron Microscopy Sciences, catalog number #12322012) following the manufacturer’s instructions. Total RNA quality number #14640) using either heat polymerization. Embedded tissues were sec- and quantity were assessed by running formaldehyde-containing agarose gel and tioned at 0.5 µm using the Leica Ultracut UCT (Leica Microsystems, Inc.) and

10 NATURE COMMUNICATIONS | (2021) 12:4941 | https://doi.org/10.1038/s41467-021-25128-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25128-y ARTICLE stained using a 0.5% Toluidine Blue O dye (Electron Microscopy Sciences, catalog These libraries were sequenced on a NextSeq 550 instrument to produce 76 nt number #26074-15). Microscopy images were captured using a ZEISS Axio Zoom. single-end reads using custom primers as described14. NanoPARE libraries were V15 microscope using the PlanNeoFluar Z 2.3×/0.57 FWD 10.6 mm objective lens trimmed of the adapter (5′-CTGTCTCTTATACACATCT-3′) using a standard with a magnification of ×260. Digital images were captured at 2584 × 1936 pixel pipeline29 and all reads were chopped to a uniform length of 20 nt, matching resolution at 12 bit/channel. standard PARE data. Finally, sPARTA was used for the trigger-target validation using default parameters for PARE data33. sRNA fluorescent in situ hybridization. The sRNA fluorescent in situ hybridi- zation (sRNA-FISH) experiment was carried out as described before48. Briefly, Data availability fresh unopened buds of strawberry were dissected and fixed in a 20 ml glass vial Data supporting the findings of this work are available within the paper and its using 4% paraformaldehyde in 1× PHEM buffer (5 mM HEPES, 60 mM PIPES, 10 Supplementary Information files. A Reporting Summary for this Article is available as a mM EGTA, 2 mM MgSO4 pH 7). Fixation was done in a vacuum chamber at 0.08 Supplementary Information file. All the sRNA, RNA-seq, and nanoPARE data generated MPa for three times, 15 min each. After fixation, samples were sent for paraffin during this study were deposited in NCBI’s SRA (Sequence Read Archive) under the embedding at histology lab from Nemours/Alfred I. duPont Hospital for Children accession number PRJNA669702. All the RNA-seq and sRNA data from public databases fi (Wilmington, DE). Samples were sectioned using a paraf n microtome and dried used for the analysis are listed in Supplementary Data 13. Source data are provided with fi on poly-L-lysine-treated coverslips. FISH hybridization was modi ed from the this paper. protocol by Javelle et al.49 by replacing the antibody with primary anti-Digoxigenin Fab fragment (Sigma-Aldrich catalog number #11214667001) and secondary donkey anti-sheep IgG (H + L) AF647, AF568, or AF633 (ThermoFisher Scientific Received: 15 October 2020; Accepted: 24 July 2021; catalog number #A-21448, A-21099, and A-21100); the dilution factors for the primary incubation were 1 : 100 in washing buffer and for secondary incubation were 1 : 1000. Briefly, samples were de-paraffinized using Histo-Clear (Fisher sci- entific, 50-899-90147) and re-hydrated by going through an ethanol series of 95%, 80%, 70%, 50%, 30%, and 10% (vol/vol) (30 s each) and through water (1 min) at References room temperature. After protease (Sigma, P5147) digestion (20 min, 37 °C), sam- 1. Fei, Q., Xia, R. & Meyers, B. C. Phased, secondary, small interfering RNAs in ples were treated with 0.2% glycine (Sigma-Aldrich, catalog number #G8898) for 2 posttranscriptional regulatory networks. Plant Cell 25, 2400–2415 (2013). min. After two washes in 1× phosphate-buffered saline, samples were dehydrated 2. Zhai, J. et al. Spatiotemporally dynamic, cell-type-dependent premeiotic and and then hybridized with probes overnight at 53.3 °C. Ten milliliters of hybridi- meiotic phasiRNAs in maize anthers. Proc. Natl Acad. Sci. USA 112, μ zation buffer contains 875 l of nuclease-free H2O, 1.25 ml in situ hybridization 3146–3151 (2015). salts, 5 ml of deionized formamide, 2.5 ml of 50% (wt/vol) dextran sulfate, 250 μlof 3. Kakrana, A. et al. Plant 24-nt reproductive phasiRNAs from intramolecular 50× Denhardt’s solution, and 125 μl of 100 mg/ml tRNA. Hybridized slides were duplex mRNAs in diverse monocots. Genome Res. 28, 1333–1344 (2018). then washed twice using 0.2× SSC buffer (saline-sodium citrate). 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Commun. 10, 627 (2019). were also hybridized to non-labeled samples as negative controls. Probes are shown in Supplementary Data 12. 10. Hollender,C.A.,Geretz,A.C.,Slovin,J.P.&Liu,Z.Flowerandearlyfruit development in a diploid strawberry, Fragaria vesca. Planta 235,1123–1139 (2012). 11. Zhang, Y., Xia, R., Kuang, H. & Meyers, B. C. The diversification of plant smFISH for phasiRNAs. Fifty probes were designed, corresponding to the top 50 NBS-LRR defense genes directs the evolution of microRNAs that target them. 21 nt phasiRNAs based on abundance in our libraries (i.e., the most abundant were Mol. Biol. Evol. 33, 2692–2705 (2016). selected). Each probe is 17–22 nt in length. The probes are designed using a web 12. Arikit, S. et al. An atlas of soybean small RNAs identifies phased siRNAs from program: Stellaris Probe Designer (https://www.biosearchtech.com/support/tools/ hundreds of coding genes. Plant Cell 26, 4584–4601 (2014). design-software/stellaris-probe-designer). 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Spectral data for pure Alexa Fluor® fluorophores were barley but not rice and maize. Plant Physiol. 184, 1407–1423 (2020). used as positive controls and non-labeled samples were used to obtain auto- 19. Patel, P. et al. Reproductive phasiRNA loci and DICER-LIKE5, but not fluorescence spectra for linear spectral unmixing. Brightness and contrast of images microRNA loci, diversified in monocotyledonous plants. Plant Physiol. 185, in the same figure panel were adjusted equally and linearly in Zen software (Carl 1764–1782 (2021). Zeiss). 20. Borges, F. et al. Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis. Nat. Genet. 50, 186–192 (2018). NanoPARE library construction, sequencing, and data analysis. NanoPARE 21. Wang, Z. et al. Polymerase IV plays a crucial role in pollen development in – libraries were constructed from 5 ng total RNA obtained from pre-meiotic stage Capsella. 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