Construction of Satellite Genetic System for Robust and Versatile Inter-species Gene Function Analyses in Viola

The genus Viola is among the largest genera in angiosperm, which includes many ornamental crops and wild herbs broadly consumed in human society. The members of Viola are one of very few taxonomic groups that produce cyclotide, a novel molecular scaffold for drug discovery, and various unique adaptive behaviors, such as dimorphic pollination strategies and mutualistic interaction with insect in seed dispersal, are also widespread across Viola species. However, the lack of effective model species that are genetically accessible and insufficiently cataloged genetic engineering methods have rendered the genus remaining largely unexplored. Here, using V. philippica as a central model organism, our study constructs the satellite genetic system that enables versatile and robust comparative gene function studies in Viola . In V. philippica , in vitro whole plant regeneration from various somatic tissues was achieved through the relayed actions of two cytokinin analogs, thidiazuron (TDZ) and 6-benzylaminopurine (BAP), and the optimized de novo plant regeneration led to highly efficient and heritable Agrobacterium -dependent genetic transformation being constructed. We further demonstrate CRISPR/Cas9-mediated site-directed genome editing that resulted in induced polymorphisms at the selected genomic loci and tobacco rattle virus-based virus-induced gene silencing (TRV-VIGS) that elicited the targeted and systemic host gene silencing, thereby presenting multiple routes to manipulating gene functions in the model Viola species. Furthermore, by applying the techniques established in V. philippica to multiple other Viola species that differ markedly in their evolutionary behaviors, the potent comparative study system was verified. Our study therefore provides a methodological framework instigating molecular dissection and engineering of novel genetic traits in phytochemistry, body architecture, and organ differentiation that evolved during the diversification of Viola species.