Generation of Low-Glutelin Rice (Oryza sativa L.) Germplasm Through Long Fragment Deletion Using CRISPR/Cas9-Mediated Targeted Mutagenesis

Rice (Oryza sativa L.) is a staple cereal crop for about half of the global population, with protein being the second-most significant nutritional component in rice grains. The storage proteins in rice grains mostly consist of glutelin, prolamin, globulin, and albumin, among which the content of easy-to-digest glutelin is the highest. Consequently, common rice increases the burden of kidney and accelerates the progression of renal disorders. The method of generating low-glutelin rice germplasm will provide novel genetic material for the cultivation of functional rice cultivars suitable for individuals with kidney diseases.

We utilized Suxiu 867 (SX867), an elite japonica rice cultivar appropriate for cultivation in Jiangsu province, as a transgenic recipient to delete a fragment of approximately 3 500 bp between the B subfamily glutelin-coding genes GluB4 and GluB5 using CRISPR/Cas9-mediated gene editing technology. The large fragment deletion was identified by PCR using the primers corresponding to the flanking sequence of gene editing target sites, while sequence-specific primers for Cas9 and hygromycin resistance gene cassettes were used to identify the low-glutelin rice mutant absent of transgenic elements. The protein component contents of homozygous low-glutelin mutants were analyzed qualitatively and quantitatively, and the expression levels of glutelin-coding genes in rice grains were detected by quantitative PCR. The agronomic traits and quality traits of homozygous low-glutelin mutants and recipient cultivar cultivated under the same cultivation conditions were measured.

Homozygous mutants with a 3 448 bp deletion between GluB4 and GluB5 genes were generated successfully. In the mutants, the relative proportion of glutelin decreased significantly, while that of prolamin and globulin increased significantly. The glutelin content of homozygous mutants decreased to 45.54%-49.75% compared to recipient cultivar, and the reduction level is comparable to LGC-1, a low-glutelin rice germplasm commonly used as a donor of low-glutelin trait in commercialized rice cultivars. The expression levels of B subfamily glutelin-coding genes in homozygous mutant were decreased significantly, and the changing trends was consistent with that of LGC-1 derived rice cultivar. Except that plant height decreased and grain length increased significantly, other measured agronomic and quality traits of homozygous mutants were not changed significantly compared to recipient cultivar.

Using CRISPR/Cas9-mediated gene editing technology, rice mutants with significant lower glutelin content free from transgenic elements were obtained successfully providing a convenient and quick method to generate low-glutelin germplasm.