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

PhasiRNAs (phased, small interfering RNAs) 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-nt and meiotic 24-nt phasiRNAs. More recently, a group of premeiotic 24-nt phasiRNAs was discovered in the anthers of two 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 three anther stages in six Pooideae species and one 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 seven species examined. These phasiRNAs exhibit distinct biogenesis mechanisms and potential Argonaute effectors compared to meiotic 24-nt phasiRNAs. We show that specific Argonaute genes co-expressed 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 three classes of phasiRNAs may be rapidly evolving or undetectable.