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

Methods

To evaluate editing results for the two candidate genes in allopolyploid T. mirus, we genotyped each homeolog separately (Supplementary Figures S2, S3). For the MYB10 gene, primers TduMYB10-F1 and TduMYB10-R1 were used to amplify the T. dubius homeolog (amplicon size: 895 bp) (Supplementary Figure S2A), and primers TpoMYB10-F1 and TpoMYB10-R1 were used to amplify the T. porrifolius homeolog (amplicon size: 936 bp) (Supplementary Figure S2B). Each 20-μL PCR reaction contained 1 μL of genomic DNA (20–100 ng), 1 × Phusion HF Buffer (New England Biolabs, Ipswich, MA, United States), 200 μM dNTPs, 0.5 μM of each primer, and 0.4 U Phusion DNA polymerase (New England Biolabs). PCR conditions for both primer sets were as follows: initial denaturation at 98 °C for 30 s; 32 cycles at 98 °C for 10 s, 61 °C annealing for 30 s, and 72 °C extension for 45 s; final extension at 72 °C for 10 min; and hold at 4 °C.

For genotyping the two homeologs of the DFR gene in T. mirus, primers Tdu-sub_DFR_F1 and Tdu-sub_DFR_R1 were used to amplify the T. dubius homeolog (amplicon size: 992 bp) (Supplementary Figure S3A), and Tpo-sub_DFR_F1 and Tpo- sub_DFR_R3 were used for the T. porrifolius homeolog (amplicon size: 1,053 bp) (Supplementary Figure S3B). The PCR setting was identical to that used for MYB10 genotyping, except the annealing temperature was adjusted to 61.5 °C and the extension time was increased to 1 min.

PCR products were sent for Sanger sequencing at Eurofins Genomics, Louisville, KY, United States. Genotypes were inferred by manually inspecting the chromatograms, following the approach described in Shan et al. (2018), with the assistance of the Synthego ICE Analysis tool (v.3; https://ice.editco.bio/#/).