Comparative RNA profiling identifies stage-specific phasiRNAs and coexpressed Argonaute genes in Bambusoideae and Pooideae species

Phased, small interfering RNAs (PhasiRNAs) play a crucial role in supporting male fertility in grasses. Earlier work in maize (Zea mays) and rice (Oryza sativa)-and subsequently many other plant species-identified premeiotic 21-nucleotide (nt) and meiotic 24-nt phasiRNAs. More recently, a group of premeiotic 24-nt phasiRNAs was discovered in the anthers of 2 Pooideae species, barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Whether premeiotic 24-nt phasiRNAs and other classes of reproductive phasiRNAs are conserved across Pooideae species remains unclear. We conducted comparative RNA profiling of 3 anther stages in 6 Pooideae species and 1 Bambusoideae species. We observed complex temporal accumulation patterns of 21-nt and 24-nt phasiRNAs in Pooideae and Bambusoideae grasses. In Bambusoideae, 21-nt phasiRNAs accumulated during meiosis, whereas 24-nt phasiRNAs were present in both premeiotic and postmeiotic stages. We identified premeiotic 24-nt phasiRNAs in all 7 species examined. These phasiRNAs exhibit distinct biogenesis mechanisms and potential Argonaute effectors compared to meiotic 24-nt phasiRNAs. We show that specific Argonaute genes coexpressed with stage-specific phasiRNAs are conserved across Bambusoideae and Pooideae species. Our degradome analysis identified a set of conserved miRNA target genes across species, while 21-nt phasiRNA targets were species-specific. Cleavage of few targets was observed for 24-nt phasiRNAs. In summary, this study demonstrates that premeiotic 24-nt phasiRNAs are present across Bambusoideae and Pooideae families, and the temporal accumulation of other classes of 21-nt and 24-nt phasiRNA differs between bamboo and Pooideae species. Furthermore, targets of the 3 classes of phasiRNAs may be rapidly evolving or undetectable.