Identifying Genes Associated with Abiotic Stress Tolerance Suitable for CRISPR/Cas9 Editing in Upland Rice Cultivars Adapted to Acid Soils

Abstract

\n Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought-prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for genome editing: SUB1A, Submergence 1A, to improve tolerance to flooding, and SPDT (SULTR3;4), SULTR-like Phosphorus Distribution Transporter, to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild-type (non-desirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system and specialized reagents and protocols were developed for SPDT editing, including vectors targeting the gene, protoplast transfection transient assays, and plant regeneration and transformation protocols by Agrobacterium and ribonucleoprotein complexes. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P-use efficiency of upland rice grown in acidic soils.