AUTHOR=May David , Sanchez Sara , Gilby Jennifer , Altpeter Fredy 

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

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1225775

DOI=10.3389/fpls.2023.1225775

ISSN=1664-462X

ABSTRACT=<p>Polyploidy is common among grasses (<italic>Poaceae</italic>) 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 (<italic>Paspalum notatum</italic> Flüggé; 2<italic>n</italic>=4<italic>x</italic>=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 (<italic>MgCh</italic>) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and <italic>npt</italic>II 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 <italic>MgCh</italic> likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. <italic>MgCh</italic> 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 <italic>MgCh</italic> 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.</p>