Blooming Diversity: Leveraging Genome Editing for Novel Diversity in Days to Flowering in Rice (Oryza sativa)

Abstract

Rice is an essential food crop providing the daily caloric intake for over 50% of the world’s population. Flowering is one of the most sensitive stages of rice growth and is highly variable among varieties and across environments. In Texas, early maturing varieties are desired as they can avoid flowering in the hottest part of the summer and allow a ratoon crop to mature before cold temperatures set in. In a previous study a diversity panel was genotyped using the 7K Cornell-IR LD Rice Array, and a genome-wide association study (GWAS) was performed using phenotypic data from the diversity panel grown in Beaumont, TX to identify genetic loci promoting early flowering in Texas germplasm. Two candidate genes, Hd3a and RFT1, underlying a major locus controlling flowering time were identified in the GWAS. In the current study, these loci were targeted for validation using multiplexed CRISPR/Cas genome editing in an elite variety from Texas (Presidio) to identify how each locus acted individually and in combination with each other. Phenotyping and mutation analysis of T0 plants was performed to correlate the effects of knocking out each of the two target loci. Plants with double knockouts at the two loci had not flowered as of 270 days, compared to 122 days to flowering for the wildtype plants. This confirmed the importance of these loci for initiation of flowering. Alongside GWAS validation, six previously identified repressors of days to flowering were simultaneously targeted for genetic knockouts using multiplexed genome editing to identify the best combination of flowering time genes for the growing environments in Texas. A range of variation was noted in the gene-edited T0 plants, with potential knockout mutations identified in five of the loci. Finally, I aimed to test a novel delivery method using the cell-penetrating peptide dfTAT to deliver Cas9 protein and a guide RNA (gRNA) into rice protoplasts as plants which have no transgenic DNA incorporated are not regulated as genetically modified organisms. Since it was difficult to determine whether the cell-penetrating peptide could deliver Cas9 inside rice protoplasts, exploring alternative delivery systems is preferred over further testing and optimization of dfTAT in rice.