Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon (Asteraceae)

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-..., 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,..., , # 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`

#...,

多倍化或全基因组复制（whole-genome duplication, WGD）是一类重要的进化驱动力。然而，调控多倍体基因组进化的分子机制仍不明晰，这一困境很大程度上源于缺乏针对最能体现WGD早期阶段的物种的功能分析工具。婆罗门参属（Tragopogon，菊科Asteraceae）是研究多倍化直接效应的经典进化模式系统。
本研究大幅优化了遗传转化体系，获得了经基因组编辑的T. porrifolius（2倍体）与T. mirus（4倍体）初代（T0）个体。研究利用CRISPR/Cas9技术，敲除了两个物种中调控花青素合成的二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase, DFR）基因。所有转基因异源四倍体T. mirus个体均至少携带1个突变DFR等位基因，其中71.4%的个体的全部4个DFR等位基因均被成功编辑。所得突变体缺失花青素，且该突变可稳定遗传至T1代。本研究证实了一套高效的CRISPR基因组编辑平台，可生成……
本数据集包含经CRISPR介导编辑的婆罗门参突变体的DFR基因测序结果。
为对T. porrifolius进行基因分型，我们使用引物TragDFR-F1与TragDFR-R2，从TpoDFR中扩增出一段包含CRISPR靶位点的DNA片段。取1 μL基因组DNA（20~100 ng）加入至20 μL PCR反应体系中，该体系包含1× Phusion HF缓冲液（New England Biolabs，美国马萨诸塞州伊普斯维奇）、200 μM dNTPs、0.5 μM上下游引物各0.5 μM、0.02 U/μL Phusion DNA聚合酶（New England Biolabs，美国马萨诸塞州伊普斯维奇）。PCR反应程序如下：98℃预变性30 s；随后98℃变性10 s、59.5℃退火30 s、72℃延伸1 min 15 s，共32个循环；最后72℃终延伸10 min，4℃保温。通过凝胶电泳对PCR产物进行检测，切下预期分子量（约1.7 kb）的条带，使用Wizard Plus SV Minipreps DNA纯化系统（Promega，美国威斯康星州麦迪逊）进行纯化。纯化后的PCR产物交由Eurofins Genomics（路易斯维尔，……）进行测序。
# 二倍体-多倍体模式系统婆罗门参属（菊科）中高效CRISPR介导基因组编辑平台的构建
[https://doi.org/10.5061/dryad.5x69p8dcs]
本数据集包含24个ab1格式文件，为来自T. porrifolius（D07）与T. mirus（D13）突变体的DFR基因Sanger测序结果。
# 数据与文件结构说明
每个T. porrifolius个体对应1个ab1格式文件，包含其DFR基因的Sanger测序结果。每个T. mirus个体对应2个ab1格式文件：一个文件包含其T. dubius同源拷贝的DFR基因Sanger测序结果，另一个文件包含其T. porrifolius同源拷贝的DFR基因测序结果。
T. porrifolius的文件名格式为：`T.porrifolius_[个体编号]_[测序引物]_[日期].ab1`
T. mirus的文件名格式为：`T.mirus_[个体编号]_[同源拷贝类型]_[测序引物]_[日期].ab1`
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