An efficient CRISPR-mediated genome editing system in diploid and polyploid Tragopogon (Asteraceae) enables functional studies of complex phenotypes and polyploid genome evolution

<p>Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force, especially in angiosperms. However, the underlying 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. <em>Tragopogon</em> (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the genetic transformation of <em>Tragopogon</em> and obtained genome-edited <em>T. porrifolius</em> (2<em>x</em>) and <em>T. mirus</em> (4<em>x</em>) primary generation (T₀) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (<em>DFR</em>) gene, which controls anthocyanin synthesis, in both <em>T. porrifolius</em> and <em>T. mirus</em>. All transgenic allotetraploid <em>T. mirus</em> individuals had at least one mutant <em>DFR</em> allele and 71.4% of the plants had all four <em>DFR</em> alleles (from both homeologs) edited, indicating a high efficiency of the CRISPR system in polyploid <em>Tragopogon</em>. The anticipated absence of the anthocyanin was observed in both leaf and floral tissues from <em>T. porrifolius</em> and <em>T. mirus</em> mutants. In addition, the mutations were inherited in the T₁ generation. This study demonstrates a highly efficient CRISPR platform producing genome-edited <em>Tragopogon</em> individuals that have successfully completed their life cycle. The approaches used and challenges faced in building the CRISPR system in <em>Tragopogon</em> provide a framework for building similar systems in other nongenetic models. Genome editing in <em>Tragopogon</em> paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, which holds enormous potential for both basic and applied research.</p>