DOI: 10.5061/dryad.5x69p8dcs
title: Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon (Asteraceae)

description: 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 nongenetic 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.
description: This dataset contains the sequencing results of the
 <em>DFR</em> gene in CRISPR-mediated
 <em>Tragopogon</em> mutants. To genotype <em>T.
 porrifolius</em>, primers TragDFR-F1 and TragDFR-R2 were used to
 amplify a fragment (containing the CRISPR target sites) from
 <em>TpoDFR</em>. One microliter of genomic DNA (20-100 ng) was
 added into a 20‐μl PCR (1× Phusion HF Buffer [New England Biolabs,
 Ipswich, MA, USA], 200 μM dNTPs, 0.5 μM of each primer, 0.02 U/μl Phusion
 DNA polymerase [New England Biolabs, Ipswich, MA, USA]). The PCR
 conditions were as follows: one cycle of denaturation at 98°C for 30 s; 32
 cycles at 98°C for 10 s, 59.5°C annealing for 30 s and 72°C extension for
 1min 15 s; one cycle at 72°C for 10 min; and hold at 4°C. The PCR product
 was accessed via gel electrophoresis; the band with the expected size
 (~1.7 kb) was excised from the gel and purified using the Wizard
 <em>Plus</em> SV Minipreps DNA Purification System (Promega,
 Madison, WI, USA). PCR products were then sequenced at Eurofins Genomics
 (Louisville, KY, USA). For <em>T.
 mirus</em>, we genotyped one homeolog at a time. Utilizing the SNP
 and indel information between the two homeologs of <em>TragDFR
 </em>in <em>T. mirus</em>, <em>GSP</em>
 (Wang et al., 2016) was used to design homeolog-specific primers:
 Tdu-sub_DFR_F1 and Tdu-sub_DFR_R1 were used to amplify the <em>T.
 dubius</em> homeolog (amplicon size: 992 bp), and Tpo-sub_DFR_F1 and
 Tpo-sub_DFR_R1 were used to amplify the <em>T.
 porrifolius</em> homeolog (amplicon size: 851 bp). To amplify each
 homeolog in <em>T. mirus</em>, the PCR conditions were the
 same as described above (genotyping <em>T. porrifolius</em>)
 except for the annealing temperature and extension time. For both sets of
 primers, the annealing temperature was 61.5°C and the PCR extension time
 was 45 s. PCR products were then purified and sequenced as described
 above.
description: # Development of an efficient CRISPR-mediated genome editing platform in
 the diploid-polyploid model system Tragopogon (Asteraceae)
 [https://doi.org/10.5061/dryad.5x69p8dcs](https://doi.org/10.5061/dryad.5x69p8dcs) This dataset (24 ab1 files) contains the Sanger sequencing results of the *DFR* gene from *T. porrifolius* (D07) and *T. mirus* (D13) mutants. ## Description of the data and file structure For each *T. porrifolius* individual, there is one ab1 file containing the Sanger sequencing result of the *DFR* gene. For each *T. mirus* individual, there are two ab1 files: one file contains the Sanger sequencing result of the *DFR* gene from the *T. dubius* homeolog, and the other file contains the sequencing result of the gene from the *T. porrifolius* homeolog. For each *T. porrifolius* file, the format is: `T.porrifolius_[individual-id]_[sequencing-primer]_[date].ab1` For each *T. mirus* file, the format is: `T.mirus_[individual-id]_[homeolog]_[sequencing-primer]_[date].ab1` ## Code/Software *Geneious* (11.0.5) was used to open the ab1 files and interpret the sequencing results.