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

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.