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

Abstract

In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3′-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants.

Pre-meiotic 21-nucleotide reproductive phasiRNAs emerged in seed plants and diversified in flowering plants

“…We found that 21 nt reproductive phasiRNAs are present in several eudicots: wild strawberry, rose, the basal eudicot columbine, and flax. Whereas previous studies have shown that 21 nt reproductive phasiRNAs exist in one gymnosperm 8 and more widely in monocots 2 , 4 , 7 , 18 , 19 (Fig. 6a ), this pathway has not been described in several well-studied eudicot families including the Brassicaceae, Fabaceae, and Solanaceae 2 , 12 , 13 .…”

The Streptochaeta Genome and the Evolution of the Grasses

“…Reproductive phasiRNAs are a subset abundant in anthers and in some cases essential to male fertility. Genomes of grass species are particularly rich in reproductive PHAS loci ( Patel et al, 2021 ), expressed in anthers but not in female reproductive tissues or vegetative tissues. Previous species studies identified hundreds of PHAS loci in anthers of maize ( Zhai et al, 2015 ) to thousands of PHAS loci in rice ( Fei et al, 2016 ), barley ( Bélanger et al, 2020 ), and bread wheat ( Bélanger et al, 2020 ; Zhang et al, 2020 ).…”

“…Total RNA was isolated using the PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, United States). 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.…”

“…The sRNA-seq data were reported in a previous study ( Patel et al, 2021 ). Also, one library of RNA-Seq (SRR3233339) used for annotation was previously published ( Givnish et al, 2010 ).…”

24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers

“…In plants, small RNAs (sRNAs) belong to three main categories: microRNAs (miRNAs) involved in target mRNA cleavage, heterochromatic small interfering RNAs (hc-siRNAs) involved in RNA-directed DNA methylation (RdDM), and phased, secondary small interfering RNAs (phasiRNAs). PhasiRNAs were initially reported in male reproductive organs of maize and rice [1][2][3][4][5][6] , and more recently in diverse monocots and eudicots [7][8][9][10] ; they are found primarily in anthers. In most species, 21-nt phasiRNAs predominate during early anther development, while 24-nt phasiRNAs are abundant during meiosis.…”