Abstract Detail

Functional Genetics/Genomics

Shan, Shengchen [1], Hauser, Bernard [2], Barbazuk, W. Brad [3], Soltis, Pamela [4], Yang, Bing [5], Soltis, Douglas [6].

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an Evolutionary Model for the Study of Polyploidy.

The genetic consequences following polyploidy (i.e., whole-genome duplication; WGD) vary greatly across organisms and through time since polyploidization. At the gene level in allopolyploids, changes include loss/retention of both parental gene copies, function/expression divergence between the two parental copies, and silencing of one parental copy. Functional studies of genes with different retention patterns contribute to a better understanding of the genetic factors underlying the success of polyploids. Most research on gene function in polyploids focuses on a few well-established genetic models or crops. However, many species that best exemplify the polyploidy process are non-genetic models; the lack of an efficient genome editing system hinders functional studies in these systems. 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 the development of robust transformation and regeneration systems in Tragopogon. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this evolutionary model system. Agrobacterium-infected leaf explants were placed on M5 medium (4.44 g/L MS salt and vitamins, 30 g/L sucrose, 1 mg/L synthetic cytokinin 6-benzylaminopurine [BAP], 0.5 mg/L synthetic auxin naphthaleneacetic acid [NAA] and 5 g/L gelrite; pH 5.8) for callus induction. The calli were then transferred to COCO-1 medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose, 5% coconut water, 2.5 mg/L BAP and 8.5 g/L agargellan; pH 5.7) and cultivated for 2 weeks. The calli were placed on WPM medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose and 8.5 g/L agargellan; pH 5.7) for shooting. The shooting rate was 33.0%. The shoots were excised from the calli and transferred to COCO-R medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose, 1.5 mg/L Indole-3-butyric acid [IBA] and 8.5 g/L agargellan; pH 5.7) for rooting with a rooting rate of 48.0%. The albino phenotype of the transgenic Tragopogon plants demonstrated that TraPDS has been knocked out by CRISPR. Successfully developing CRISPR in Tragopogon holds enormous potential for examining the biological underpinning of various gene retention patterns following WGD in this system. We hope our studies and methods developed for Tragopogon will provide a guideline for establishing a CRISPR system in other non-genetic model polyploids of evolutionary or other interest.

1 - Florida Museum of Natural History, 1659 Museum Rd, Gainesville, FL, 32611, United States
2 - Department Of Botany, 220 Bartram Hall, PO Box 118526, Gainesville, FL, 32611, United States
3 - Department of Biology, University of Florida
4 - University Of Florida, Florida Museum Of Natural History, Gainesville, FL, 32611.0, United States
5 - University of Missouri, Donald Danforth Plant Science Center
6 - University of Florida, Florida Museum of Natural History,, 3215 Hull Road, P. O. Box 2710, Gainesville, FL, 32611, USA

none specified

Presentation Type: Oral Paper
Number: FGG1001
Abstract ID:605
Candidate for Awards:Margaret Menzel Award

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