Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon...

Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force. However, the mechanisms governing polyploid genome evolution remain unclear, limited largely by a lack of functional analysis tools in organisms that best exemplify the earliest stages of WGD. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the transformation system and obtained genome-edited T. porrifolius (2x) and T. mirus (4x) primary generation (T0) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (DFR) gene, which controls anthocyanin synthesis, in both species. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele, and 71.4% had all four DFR alleles edited. The resulting mutants lacked anthocyanin, and these mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform, producing genome-edited Tragopogon individuals that have completed the life cycle. The approaches used and challenges faced in building the CRISPR system in Tragopogon provide a framework for building similar systems in other non-genetic models. Genome editing in Tragopogon paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, holding enormous potential for both basic and applied research.