Abstract Detail

Functional Genetics/Genomics

Shan, Shengchen [1], Yang, Bing [2], Hauser, Bernard [3], Mavrodiev, Evgeny [4], Soltis, Pamela S. [5], Soltis, Douglas [6].

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

Recently formed natural polyploid species of Tragopogon (Asteraceae) provide an excellent model system for studying the immediate consequences of polyploidization. Application of CRISPR/Cas9, an efficient and customizable genome editing technology, to Tragopogon will facilitate novel studies of the genetic consequences following polyploidization. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Half-strength MS medium with 0.5 mg/L NAA and 1.0 mg/L BAP was used for callus induction from cotyledon explants. MS medium with 0.4 mg/L BAP was adopted for shoot regeneration. Roots were regenerated from the shoot apex on half-strength MS medium with 0.05 mg/L NAA. Hygromycin B was found to suppress callus growth and shoot regeneration. Agrobacterium-mediated transformation of Tragopogon cotyledon explants produced transgenic calli at a high efficiency. We also established an efficient protocol for Tragopogon protoplast isolation and transfection. Use of a CRISPR/Cas9 system, built upon Arabidopsis ubiquitin promoter and U6 promoters for Cas9 and single guide RNA (sgRNA) expression, respectively, was capable of introducing site-specific mutations in Tragopogon protoplasts, through editing the exogenous nonfunctional GFP gene for gain of green fluorescence signal. Approximately 6.8% of the protoplasts displayed strong GFP signal 60 h after transfection. Finally, to test the feasibility of utilizing CRISPR/Cas9 for endogenous gene editing in Tragopogon, Agrobacterium-mediated transformation with Cas9/sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sanger sequencing indicated simultaneous mutation of two copies and four copies of TraPDS in albino shoots from T. porrifolius (2x) and T. mirus (4x), respectively. The proportion of successfully transformed calli with the albino phenotype reached on average 87% and 78% in the diploid and polyploid, respectively. To our knowledge, this is the first application of CRISPR/Cas9 in any natural polyploid system, and one of the few cases of CRISPR/Cas9-mediated mutation in species of Asteraceae. This work illustrates the potential of applying CRISPR/Cas9 technology in studies of evolution. Potential applications of a usable CRISPR/Cas9 system will permit unique studies of genome dominance in polyploids and investigation of those genes involved in phenotypic changes, such as distinct inflorescence morphologies in reciprocally formed polyploid T. miscellus.

1 - Florida Museum of Natural History, Dickinson Hall, 1659 Museum Rd, Gainesville, FL, 32611, USA
2 - Genetics, Development and Cell Biology, 1035C Roy J. Carver Co-Lab, Ames, IA, 50011, USA
3 - Department of Biology, 516A Bartram Hall, Gainesville, FL, 32611, USA
4 - Florida Natural History Museum, Florida Museum Of Natural History PO Box 117800, Gainesville, FL, 32611, United States
5 - University Of Florida, Florida Museum Of Natural History, Po Box 117800, Gainesville, FL, 32611, United States
6 - University of Florida, Biology, Gainesville, FL

Keywords:
Polyploidy
CRISPR/Cas9
Tragopogon.

Presentation Type: Oral Paper
Session: 8, Functional Genetics and Genomics
Location: 109/Mayo Civic Center
Date: Monday, July 23rd, 2018
Time: 8:45 AM
Number: 8002
Abstract ID:321
Candidate for Awards:Margaret Menzel Award