Development of a homeolog-specific gene editing system in an evolutionary model for the study of polyploidy in nature

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. <i>Tragopogon</i> (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 allotetraploid <i>T. mirus</i>. Using the <i>MYB10</i> and <i>DFR</i> genes as examples, we successfully knocked out the targeted homeolog in <i>T. mirus</i> (4<i>x</i>) 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% for <i>MYB10</i> and <i>DFR</i>, respectively. Biallelic modification of the targeted homeolog occurred in the T₀ generation. These results demonstrate the robustness of homeolog-specific editing in polyploid <i>Tragopogon</i>, laying the foundation for future studies of genome evolution following WGD in nature.