A novel method of generating RNAi libraries for high‐throughput gene function analysis of creeping bentgrass

Abstract

RNA interference (RNAi) is a powerful tool for gene function analysis, particularly for outcrossing polyploid species where simultaneous mutations of multiple alleles of a gene with similar but nonidentical sequences may not be readily achieved with the novel clustered regularly interspaced short palindromic repeats/Cas9 gene editing tools. The most effective RNAi strategy in plants is to use long hairpin RNAi (lhRNAi) constructs containing inverted repeats (IRs) of target genes. Previously, we used a Phi29-Amplified RNAi Construct method to create a single lhRNAi construct that was shown to be highly effective in gene silencing in the model grass Brachypodium distachyon (L.) P.Beauv. Here, we describe a method of creating a library of lhRNAi constructs ready for high-throughput gene function analysis in creeping bentgrass (Agrostis stolonifera L.), an important turfgrass species. A cDNA population in which salt-responsive genes were enriched by suppression subtractive hybridization was used as the source of IRs. Sequencing of 13 randomly selected colonies from the lhRNAi library showed that the size of IRs varied from 184 to 994 bp, which is appropriate for RNAi, and nine of the IRs were shown to be homologous to known genes in closely related grasses. In addition to the demonstration of the proof-of-concept of creating an expression-ready library, the salt stress-specific lhRNAi library represents a valuable resource for studying the functions of salt-responsive genes in creeping bentgrass. The method described here is broadly applicable to all plant species where an efficient plant transformation platform is available.