Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9

David May; Sara Sanchez; Jennifer Gilby; Fredy Altpeter

doi:10.3389/fpls.2023.1225775

Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9

Front Plant Sci. 2023 Jul 13:14:1225775. doi: 10.3389/fpls.2023.1225775. eCollection 2023.

Authors

David May¹, Sara Sanchez¹, Jennifer Gilby¹, Fredy Altpeter^{1

2

3}

Affiliations

¹ Agronomy Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States.
² Genetics Institute, University of Florida, Gainesville, FL, United States.
³ Plant Cellular and Molecular Biology Program, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States.

Abstract

Polyploidy is common among grasses (Poaceae) and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass (Paspalum notatum Flüggé; 2n=4x=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf. The chlorophyll biosynthesis gene magnesium chelatase (MgCh) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and nptII were delivered to callus cultures by biolistics. Edited plants were characterized through PCR-based assays and DNA sequencing, and mutagenesis frequencies as high as 99% of Illumina reads were observed. Sequencing of wild type (WT) bahiagrass revealed a high level of sequence variation in MgCh likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. MgCh mutants exhibited visible chlorophyll depletion with up to 82% reductions in leaf greenness. Two lines displayed progression of editing over time which was linked to somatic editing. Apomictic progeny of a chimeric MgCh editing event were obtained and allowed identification of uniformly edited progeny plants among a range of chlorophyll depletion phenotypes. Sanger sequencing of a highly edited mutant revealed elevated frequency of a WT allele, probably due to frequent homology-directed repair (HDR). To our knowledge these experiments comprise the first report of genome editing applied in perennial, warm-season turf or forage grasses. This technology will accelerate bahiagrass cultivar development.

Keywords: CRISPR/Cas; bahiagrass; forages; gene editing; homology-directed repair; polyploidy; targeted mutagenesis; turf.

Grants and funding

This research was co-funded by the University of Florida and the USDA National Institute of Food and Agriculture, Hatch project 1020425. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the University of Florida or USDA National Institute of Food and Agriculture.