Removing Harmful Pericarp Character of Weedy Rice as the First Step of Domestication towards Direct-Seeding Rice Using CRISPR/Cas9-Targeted Mutagenesis

Abstract

With the scarcity of fresh water resources and the increase in labor prices, rice cultivation is changing from transplanting to direct seeding. Weedy rice is a malignant weed with strong drought tolerance in seed germination in direct-seeding rice fields. As the same species of cultivated rice (Oryza sativa L.), weedy rice (Oryza sativa f. spontanea) has the potential of domestication into direct-seeding rice with strong drought tolerance in seed germination by changing a few unfavorable traits. The red pericarp, one of the harmful traits of weedy rice, seriously affects the quality and commercial value of cultivated rice. The recently developed CRISPR/Cas9 (Clustered Regular Interspaced Short Palindromic Repeats) technology can accurately edit the function of genes, providing a possibility for the directed evolution. Weedy rice (WRL-162) with red pericarp was used as a mutant material. Two mutation sites, upstream and downstream of the 14 bp differential fragment of exon 6 of Rc gene, were designed using the CRISPR-Cas9 gene-editing technique. Consequently, three mutant types in T1 generation weedy rice with CRISPR-Cas9 vector changed from red to white pericarp. One mutant type in T1 generation weedy rice with CRISPR-Cas9 vector maintained red pericarp. Comparing the wild type of weedy rice and rice variety control (Nipponbare), mutant types in T3 generation weedy rice without CRISPR-Cas9 vector significantly improved the drought resistance in seed germination. In addition, there was no significant difference in panicle number, seed setting rate per plant, grain length and width between wild type and T3 generation mutant weedy rice without CRISPR-Cas9 vector. Comparing the wild type, some mutant types in T3 generation weedy rice without CRISPR-Cas9 vector significantly decreased plant height, decreased spikelet number per main panicle, decreased plump seed number per main panicle, and increased 1000-grain weight. Our results showed that the Rc gene editing using CRISPR/Cas9 technology can not only remove the harmful pericarp character of weedy rice, but also improve drought tolerance in seed germination. This study might provide new insights for the utilization of weedy rice as germplasm resources for direct-seeding rice by precisely editing genes to remove unfavorable traits.