Accelerated Breeding of Cereal Crops
Citation
Schernthaner, J., Balcerzak, M., Murmu, J., Subramaniam, R. (2022). A Genotyping Protocol to Identify CRISPR/Cas9-Edited Events in Hexaploid Wheat. In: Bilichak, A., Laurie, J.D. (eds) Accelerated Breeding of Cereal Crops. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1526-3_12
Plain language summary
We present a protocol for analyzing transgenic wheat plants in which we changed a gene using the Crispr/Cas9 molecular tools. The protocol addresses the technical challenges that occur when analyzing transgenic plants with more than one genome as is the case in bread wheat. Presented is a technical solution to simplify the selection of plants which carry the wanted introduced mutations.
Abstract
Targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become an established method to introduce changes into a plant genome. While traditional genetic modification allows us to follow transgenic events with selection markers as they are often co-integrated, this is not always feasible when Cas9/guide RNA-based methods are applied. In cases where genome editing does not result in a selectable phenotype, the only option to analyze editing events is by sequencing the target region through either conventional or high-throughput sequencing, both of which can be tedious and costly. In this chapter, we present a rapid, economic genotyping method that was designed to identify editing events in the three wheat sub-genomes. The method is not only economical but is in general very useful when dealing with polyploid organisms where identification and characterization of edited events can be challenging.