Reproductive phasiRNA loci and DICER-LIKE5, but not microRNA loci, diversified in monocotyledonous plants

Abstract

200 words) 1 In monocots other than maize and rice, the repertoire and diversity of microRNAs (miRNAs) and 2 the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly 3 characterized. To remedy this, we sequenced small RNAs from vegetative and dissected 4 inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging 5 angiosperm lineages, as well as publicly available data from 10 additional monocot species. We 6 annotated miRNAs, siRNAs and phasiRNAs across the monocot phylogeny, identifying miRNAs 7 apparently lost or gained in the grasses relative to other monocot families, as well as a number 8 of tRNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these 9 misannotations, we identified conservation at the 8 th , 9 th , 19 th and 3' end positions that we 10 hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show 11 that 21-nt reproductive phasiRNAs are far more numerous in grass genomes than other 12 monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5 (DCL5), 13 important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the 14 diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, 15 siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued 16 exploration of small RNA diversification in flowering plants. 17 18 19 20 55 2018). Rice, Brachypodium, and maize are the most studied of the grasses, with miRNAs 56 characterized using varying genotypes, tissue types, growth and stress conditions (Jeong et al., 57 2011; Zhang et al., 2009). With the major goal of assessing the diversity and origins of miRNAs 58 4in monocots, we analyzed sRNA data from 38 phylogenetically diverse monocots, spanning 59 orders from the Acorales to the Zingiberales. We described sRNA size classes, miRNA 60 conservation, divergence, sequence variability, 5' and 3' end nucleotide preferences, and single 61 nucleotide sequence profile characterizing positional biases and providing novel insights within 62 the miRNA sequences. We performed comparative analysis of miR2118 and miR2275 and their 63 long non-coding RNA (lncRNA) targets in monocots relative to other flowering plants, 64 demonstrating their presence and absence in these species. We found that both miR2118 and 65 miR2275 are conserved across diverse monocot species, and are present in vegetative tissues but 66 are found at high abundances predominantly in inflorescence tissues. The 21-and 24-nt PHAS 67 loci are most numerous in the genomes of grasses, relative to other monocots, and are similarly 68 most abundant in inflorescence tissues. Fewer PHAS loci were identified in non-grass monocots. 69 Overall, our study provides a deep comparative analysis of sRNAs in monocots, including a refined 70 database of monocot miRNAs. 71 72 Results 73 74Sequencing from diverse monocots demonstrates atypically abundant 22-nt siRNAs 75 We c...

Unusual predominance of maintenance DNA methylation in Spirodela polyrhiza

“…CG methylation is present at small clusters of Long Terminal Repeat (LTR) retrotransposons in the genome ( Figure 1D ), but CHH methylation which is normally enriched in repetitive elements like LTR retrotransposons (27) , is absent ( Figure 1C DNA methylation at some CHH sites is guided by small RNAs (sRNAs) generated through the RdDM pathway (28) ( Figure 2A ), so small RNA (sRNA) sequence reads were generated for both S. polyrhiza lines to test for functional defects in the pathway ( Supplemental Table 2 ). Both lines display a distinct lack of 24-nucleotide (nt), heterochromatic siRNAs (het-siRNAs) which are typically the most abundant size class of angiosperm sRNAs (29,30) ( Figure 2B ). The highly conserved, canonical RdDM pathway produces these 24 nt het-siRNAs via DICER-LIKE 3 (DCL3) processing of an RNA Polymerase IV (Pol IV)-derived double-stranded RNA (31,32) .…”

Conserved and non-conserved triggers of 24-nt reproductive phasiRNAs in eudicots

“…This was based in part on the presence/absence of a canonical pathway trigger (miR2275). The basal angiosperms Amborella trichopoda and Nymphaea colorata are the only species outside of eudicots and monocots that have the trigger encoded in their genomes but lack bona fide 24-PHAS loci (Xia et al, 2019; Patel et al, 2020). Our confirmation of the presence of this pathway in columbine, an early diverging lineage of the eudicots, provides strong support for the emergence of the pathway prior to the emergence of the core eudicots.…”

“…The 21-nt phasiRNAs are produced by the action of DCL4 while DCL3 and DCL5 are responsible for 24-nt phasiRNA production in eudicots and monocots, respectively. DCL5 resulted from a monocot-specific duplication of DCL3 and is specialized for the production of reproductive 24-nt phasiRNAs in monocots (Teng et al, 2020; Patel et al, 2020). Although DCL3 is single copy in the genome of many eudicots, some eudicot lineages that have a duplicated copy of DCL3 are also predicted or validated for the presence of 24-nt phasiRNAs, perhaps consistent with a role for the duplicated DCL3 copy in the biogenesis of reproductive 24-nt phasiRNAs (Pokhrel et al, 2020; Xia et al, 2019).…”

“…Reproductive 24-nt phasiRNAs were reported mainly in the male organs of grasses (Poaceae) (Zhai et al, 2015b; Song et al, 2012; Johnson et al, 2009; Patel et al, 2020), and more recently in six non-grass monocots (Patel et al, 2020) and eight eudicots (Xia et al, 2019). This size class is abundant in the meiotic stage of anthers and are mainly triggered by miR2275, although there are cases of trigger-less 24-nt phasiRNA-producing loci (aka “24- PHAS ” loci) in tomato and petunia (Xia et al, 2019), and in monocots outside the Poaceae (Kakrana et al, 2018; Patel et al, 2020). The spatiotemporal pattern of 24-nt phasiRNAs in male reproductive tissues has been well-described in maize, rice, and litchi (Zhai et al, 2015b; Song et al, 2012; Xia et al, 2019).…”

TheStreptochaetagenome and the evolution of the grasses

“…Total RNA was isolated using the PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, USA). sRNA libraries were published previously (Patel et al, 2021). RNA sequencing libraries were prepared from the same material using the Illumina TruSeq stranded RNA-seq preparation kit (Illumina Inc., United States) following manufacturer’s instructions.…”