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Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs–named premeiotic 24-nt phasiRNAs–have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediatingPHASprecursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known. 

Abstract Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways. 

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia , one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions. 

Abstract The Solanaceae or “nightshade” family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family’s small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities. 

Abstract Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs 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.