Location: Plant Science ResearchTitle: Targeted mutagenesis of alfalfa
|Dornbusch, Melinda - Mindy|
|FARMER, ANDREW - National Center For Genome Resources|
|GUTIERREZ-GONZALEZ, JUAN - University Of Leon|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 5/16/2021
Publication Date: 7/18/2021
Citation: Curtin, S.J., Miller, S.S., Dornbusch, M.R., Farmer, A., Gutierrez-Gonzalez, J. 2021. Targeted mutagenesis of alfalfa. In: Yu, X. and Kole, C., editors. The Alfalfa Genome, Compendium of Plant Genomics. Cham, Switzerland: Springer. p. 271-283.
Interpretive Summary: This a methods chapter for the alfalfa research community that guides the reader in constructing reagents for targeted mutagenesis of alfalfa genes. In addition, the protocol details a procedure to screen mutant plants fast and effectively using Pacbio amplicon sequencing.
Technical Abstract: Medicago sativa (alfalfa) is a perennial outcrossing tetraploid flowering legume plant and the third most widely grown crop in the US, approximately 80 million acres. Alfalfa is an important cover and forage crop used for grazing, hay and silage in many parts of the world and is the main forage crop for meat and milk production. Not surprising, there is interest in the use of new breeding technologies (NBT) including gene editing to improve various traits such as yield and abiotic and biotic stress responses. The CRISPR/Cas9 is a highly effective gene editing reagent that can generate site-directed double-stranded breaks (DSB) resulting in frame-shift mutations. In this chapter, we describe the construction of a CRISPR/Cas9 reagent that target the Pho2 genes. Plants recovered from tissue culture were first screened for targeted mutations using combinations of the cleaved amplified polymorphic sequences (CAPS) or the target amplicon clone and sequencing assays (TACAS). These techniques are sufficient for screening targets in diploid plants but are less effective at screening multiple tetraploid targets. Therefore, PacBio® long amplicon sequencing was evaluated for mutant characterization in alfalfa. We found the platform to be rapid and accurate at characterizing multiple targets across all four haplo-alleles in single sequencing run. The raw sequencing data was processed using the SMRT link platform and analyzed using a workflow on the Geneious® software. Using these tools, hundreds of plants could be rapidly genotyped and in the transgenic plants identified, approximately 57% exhibited targeted mutations.