Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy

Abstract

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9-based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site-specific mutations in Tragopogon protoplasts. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9-based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

Transcriptome Dynamics of the Inflorescence in Reciprocally Formed Allopolyploid Tragopogon miscellus (Asteraceae)

“…Our hypothesis requires further study — an effect of pectin metabolism on inflorescence development has not been reported in Asteraceae. With an established CRISPR/Cas9 system in Tragopogon ( Shan et al, 2018 , 2020 ), the functions of related genes (such as genes encoding pectinesterase and polygalacturonase) could be rigorously examined.…”

Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

“…In this work, we successfully applied the CRISPR/Cas9 technology for the first time in chicory. CRISPR/Cas9 technology was already successfully used to knock out genes in other plants of the Asteraceae family such as salsify [40], lettuce [41] and dandelion [30]. Asteraceae as well as members of the Brassicaceae and Solanaceae are amenable to this biotechnological approach because of their high ability to regenerate [42].…”

Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification

“…The thermoinhibition gene NCED4 was successfully edited using CRISPR/Cas9 in diploid, self-compatible lettuce ( Lactuca sativa ) with editing stability and Cas9 segregation demonstrated in the next seed generation [ 24 ]. In both sexual diploid Tragopogon porrifolius and tetraploid Tragopogon mirus, a low-efficiency Agrobacterium -mediated transformation system was developed to examine CRISPR/Cas9 gene editing efficiency in T0 calli and regenerated shoots, however plants were not taken to the next generation [ 25 ]. In hexaploid self-incompatible Chrysanthemum morifolium, low-efficiency editing of an introduced fluorescent marker gene was observed [ 26 ].…”

“…Figure S5: Detection of the Cas9 transgene in 78/79 T1 progeny from T0 chimeric HPDS mutants. References [ 48 , 49 , 50 ] are cited in the supplementary materials.…”