Design of artificial miRNA for redundant silencing of Brassica SHP1 and SHP2: transient assay-based validation of transcript cleavage from polyploid Brassicas

Asynchronous pod-shatter results in significant yield losses in canola. SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) regulate pod shatter in Brassicaceae. This study describes the potential of an artificial RNA (amiRNA)-based approach for redundant silencing of SHP1/SHP2 homologs in Brassicas for introducing shatter resistance. After surveying the natural variation in Brassica SHP homologs, amiR-bnashp1 and amiR-bnashp2 were designed and processed from miR319a precursor in N. benthamiana. Analysis of thermodynamic interaction between amiRNAs and SHP1/SHP2 homologs predicted high binding energy for amiR-bnashp2 [total free energy of binding (TFEB): −23.54 kcal/mol] and not for amiR-bnashp1 (TFEB: 6.01 kcal/mol). As predicted, agro-co-infiltration assay validated amiR-bnashp2-mediated cleavage of SHP1 and SHP2 homologs from B. napus, B. juncea and A. thaliana, while amiR-bnashp1 failed to direct cleavage of these homologs. In conclusion, for trait manipulation in polyploid genomes deployment of amiRNAs is suggested to silence redundant genes. Furthermore, an a priori knowledge on transcriptome potential of genome concerned is a prerequisite for predicting efficiency and specificity of target selection by amiRNA(s).