Location: Vegetable Crops ResearchTitle: Targeted mutagenesis using CRISPR/Cas in inbred potatoes
|BUTLER, NATHANIEL - UNIVERSITY OF WISCONSIN|
|JIANG, JIMING - UNIVERSITY OF WISCONSIN|
Submitted to: American Journal of Potato Research
Publication Type: Proceedings
Publication Acceptance Date: 4/7/2017
Publication Date: N/A
Technical Abstract: Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clonal propagation. The use of an autotetraploid-based system has slowed the progress of potato breeding and genetics because it prevents fixing of important traits, limits genetic gains, and complicates genetic mapping efforts. The development of inbred potatoes using diploid (2n = 2x = 24) germplasm has provided a new tool to identify and fix important traits, and conduct functional genomics in potato. The potato clone, M18 is an S5 inbred line derived from the diploid S. tuberosum clone, US-W4. M18 is compact, self-fertile and produces round white tubers. These characteristics in addition to its near-homozygous nature make M18 an excellent candidate for conducting functional genomics in potato. A plant transformation pipeline has been developed using M18 to deliver sequence-specific nuclease (SSN) reagents, such as CRISPR/Cas and improve the efficiency of stable targeted mutagenesis. The target gene, PHYTOENE DESATURASE (PDS) was chosen for mutagenesis due to its clear phenotype and single-copy nature. Reagents for conducting CRISPR/Cas have also been optimized using this transformation pipeline and used for whole-plant transformation. Resulting events with targeted mutations, express a photo bleaching phenotype associated with PDS loss-of-function and are being self-pollinated to fix mutations and determine heritability.