Developing a CRISPR System in Nongenetic Model Polyploids

Abstract

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 functions to date focuses on a few well-established genetic models or crops. However, many species that best exemplify the polyploidy process are nongenetic models; the lack of an efficient genome editing system hinders functional studies in these systems. In this chapter, we discuss the considerations of developing CRISPR, a robust and efficient genome editing system, in polyploid plants that are not genetic models. We use diploid and polyploid Tragopogon (Asteraceae) as examples of a well-studied evolutionary model system for which abundant genetic and genomic resources are lacking. Using this system, we provide our protocols for sgRNA design, plasmid construction, a useful protoplast transient assay, and a plant transformation method we developed for this system. We also provide suggestions for possible modifications to these protocols to help promote successful application to other non-models. With the rapid applications of CRISPR in plant sciences, the broad adaptation of CRISPR in studies of the evolutionary significance of WGD holds enormous potential. We hope our studies and methods developed for polyploid Tragopogon will provide a guideline for establishing a CRISPR system in other nongenetic model polyploids of evolutionary or other interest.

Keywords: CRISPR; Genome editing; Nongenetic model; Plasmid construction; Polyploidy; Transformation; Transient assay.