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Polyploidy, or whole-genome duplication (WGD), is a significant evolutionary force. Following allopolyploidy, duplicate gene copies (homeologs) have divergent evolutionary trajectories: some genes are preferentially retained in duplicate, while others tend to revert to single-copy status. Examining the effect of homeolog loss (i.e., changes in gene dosage) on associated phenotypes is essential for unraveling the genetic mechanisms underlying polyploid genome evolution. However, homeolog-specific editing has been demonstrated in only a few crop species and remains unexplored beyond agricultural applications.Tragopogon(Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy in nature. In this study, we developed a CRISPR-mediated homeolog-specific editing platform in allotetraploidT. mirus. Using theMYB10andDFRgenes as examples, we successfully knocked out the targeted homeolog inT. mirus(4x) without editing the other homeolog (i.e., no off-target events). The editing efficiencies, defined as the percentage of plants with at least one allele of the targeted homeolog modified, were 35.7% and 45.5% forMYB10andDFR, respectively. Biallelic modification of the targeted homeolog occurred in the T₀generation. These results demonstrate the robustness of homeolog-specific editing in polyploidTragopogon, laying the foundation for future studies of genome evolution following WGD in nature. 

Abstract 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.