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An efficient CRISPR-mediated genome editing system in diploid and polyploid Tragopogon (Asteraceae) enables functional studies of complex phenotypes and polyploid genome evolution

Cite this dataset

Shan, Shengchen et al. (2024). An efficient CRISPR-mediated genome editing system in diploid and polyploid Tragopogon (Asteraceae) enables functional studies of complex phenotypes and polyploid genome evolution [Dataset]. Dryad. https://doi.org/10.5061/dryad.5x69p8dcs

Abstract

Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force, especially in angiosperms. However, the underlying 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 genetic transformation of Tragopogon 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 T. porrifolius and T. mirus. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele and 71.4% of the plants had all four DFR alleles (from both homeologs) edited, indicating a high efficiency of the CRISPR system in polyploid Tragopogon. The anticipated absence of the anthocyanin was observed in both leaf and floral tissues from T. porrifolius and T. mirus mutants. In addition, the mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform producing genome-edited Tragopogon individuals that have successfully completed their 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, which holds enormous potential for both basic and applied research.

Methods

This dataset contains the sequencing results of the DFR gene in CRISPR-mediated Tragopogon mutants. To genotype T. porrifolius, primers TragDFR-F1 and TragDFR-R2 were used to amplify a fragment (containing the CRISPR target sites) from TpoDFR. 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 Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). PCR products were then sequenced at Eurofins Genomics (Louisville, KY, USA).

For T. mirus, we genotyped one homeolog at a time. Utilizing the SNP and indel information between the two homeologs of TragDFR in T. mirus, GSP (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 T. dubius homeolog (amplicon size: 992 bp), and Tpo-sub_DFR_F1 and Tpo-sub_DFR_R1 were used to amplify the T. porrifolius homeolog (amplicon size: 851 bp). To amplify each homeolog in T. mirus, the PCR conditions were the same as described above (genotyping T. porrifolius) 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.

Funding

National Science Foundation, Award: IOS-1923234

National Science Foundation, Award: DEB-2043478