DOI: 10.5061/dryad.0vt4b8h9t
title: Development of a homeolog-specific gene editing system in an evolutionary model for the study of polyploidy in nature

description: 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.
description: To evaluate editing results
 for the two candidate genes in allopolyploid <em>T.
 mirus</em>, we genotyped each homeolog separately (Supplementary
 Figures S2, S3). For the <em>MYB10</em> gene, primers
 TduMYB10-F1 and TduMYB10-R1 were used to amplify the <em>T.
 dubius</em> homeolog (amplicon size: 895 bp) (Supplementary Figure
 S2A), and primers TpoMYB10-F1 and TpoMYB10-R1 were used to amplify the
 <em>T. porrifolius</em> 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 <em>DFR</em>
 gene in <em>T. mirus</em>, primers Tdu-sub_DFR_F1 and
 Tdu-sub_DFR_R1 were used to amplify the <em>T. dubius</em>
 homeolog (amplicon size: 992 bp) (Supplementary Figure S3A), and
 Tpo-sub_DFR_F1 and Tpo- sub_DFR_R3 were used for the <em>T.
 porrifolius</em> homeolog (amplicon size: 1,053 bp) (Supplementary
 Figure S3B). The PCR setting was identical to that used for
 <em>MYB10</em> 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/#/).
description: # Development of a homeolog-specific gene editing system in an
 evolutionary model for the study of polyploidy in nature Dataset DOI:
 [10.5061/dryad.0vt4b8h9t](10.5061/dryad.0vt4b8h9t) ## Description of the
 data and file structure PCR products were sent for Sanger sequencing at
 Eurofins Genomics, Louisville, KY, USA. 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/#/](https://ice.editco.bio/#/)).  ### Files
 and variables #### File:
 T.mirus_M01-2_Tdu-homeolog_TduMYB10F1_12122024.ab1 **Description:** Sanger
 sequencing result of the *T. dubius* *MYB10* homeolog from *T. mirus*
 individual M01-2 #### File:
 T.mirus_M01-4_Tdu-homeolog_TduMYB10F1_09122024.ab1 **Description:** Sanger
 sequencing result of the *T. dubius* *MYB10* homeolog from *T. mirus*
 individual M01-4 #### File:
 T.mirus_M01-6_Tdu-homeolog_TduMYB10F1_12122024.ab1 **Description:** Sanger
 sequencing result of the *T. dubius* *MYB10* homeolog from *T. mirus*
 individual M01-6 #### File:
 T.mirus_M01-10_Tdu-homeolog_TduMYB10F1_12122024.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *MYB10*
 homeolog from *T. mirus* individual M01-10 #### File:
 T.mirus_M01-12_Tdu-homeolog_TduMYB10F1_12122024.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *MYB10*
 homeolog from *T. mirus* individual M01-12 #### File:
 T.mirus_M01-2_Tpo-homeolog_TpoMYB10R1_12122024.ab1 **Description:** Sanger
 sequencing result of the *T. porrifolius* *MYB10* homeolog from *T. mirus*
 individual M01-2 #### File:
 T.mirus_M01-4_Tpo-homeolog_TpoMYB10R1_09172024.ab1 **Description:** Sanger
 sequencing result of the *T. porrifolius* *MYB10* homeolog from *T. mirus*
 individual M01-4 #### File:
 T.mirus_M01-10_Tpo-homeolog_TpoMYB10R1_12122024.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *MYB10*
 homeolog from *T. mirus* individual M01-10 #### File:
 T.mirus_M01-6_Tpo-homeolog_TpoMYB10R1_12122024.ab1 **Description:** Sanger
 sequencing result of the *T. porrifolius* *MYB10* homeolog from *T. mirus*
 individual M01-6 #### File:
 T.mirus_M01-12_Tpo-homeolog_TpoMYB10R1_12122024.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *MYB10*
 homeolog from *T. mirus* individual M01-12 #### File:
 T.mirus_DP-3_Tdu-homeolog_Tdu_sub_DFR_R1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *DFR*
 homeolog from *T. mirus* individual DP-3 #### File:
 T.mirus_DP-4_Tdu-homeolog_Tdu_sub_DFR_R1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *DFR*
 homeolog from *T. mirus* individual DP-4 #### File:
 T.mirus_DP-5_Tdu-homeolog_Tdu_sub_DFR_R1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *DFR*
 homeolog from *T. mirus* individual DP-5 #### File:
 T.mirus_DP-3_Tpo-homeolog_Tpo_sub_DFR_F1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *DFR*
 homeolog from *T. mirus* individual DP-3 #### File:
 T.mirus_DP-7_Tpo-homeolog_Tpo_sub_DFR_R3_01202025.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *DFR*
 homeolog from *T. mirus* individual DP-7 #### File:
 T.mirus_DP-8_Tdu-homeolog_Tdu_sub_DFR_F1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *DFR*
 homeolog from *T. mirus* individual DP-8 #### File:
 T.mirus_DP-4_Tpo-homeolog_Tpo_sub_DFR_R3_01202025.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *DFR*
 homeolog from *T. mirus* individual DP-4 #### File:
 T.mirus_DP-8_Tpo-homeolog_Tpo_sub_DFR_R3_01202025.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *DFR*
 homeolog from *T. mirus* individual DP-8 #### File:
 T.mirus_DP-5_Tpo-homeolog_Tpo_sub_DFR_R3_01202025.ab1
 **Description:** Sanger sequencing result of the *T. porrifolius* *DFR*
 homeolog from *T. mirus* individual DP-5 #### File:
 T.mirus_DP-7_Tdu-homeolog_Tdu_sub_DFR_R1_01202025.ab1
 **Description:** Sanger sequencing result of the *T. dubius* *DFR*
 homeolog from *T. mirus* individual DP-7 ## Code/software The files can be
 opened in Geneious or with [Finch
 TV](https://digitalworldbiology.com/FinchTV)