Traits improvement of wild rice O. rufipogon via multiplex genome editing

Chang Tian, Xu Tang, Guangzhong Zhang, Rui Zhang, Xinruolan Yang, Lei Ding, Caiyun Yin, Hongfei Lin, Fenglin Su, Suikang Wang, Xiaoxia Li, Lianguang Shang, Yong Zhang, Quan Wang

Journal of Integrative Plant Biology; 18 November 2025; https://doi.org/10.1111/jipb.70087

Multiplex genome editing of seven genes for the rapid improvement of target traits in wild rice Oryza rufipogon produced lines with enhanced agronomic traits, including erect plant architecture, shortened awns, yellowish hull color, reduced seed shattering, white-colored pericarps, and other improvements.

Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives ~80% of its 993 identified domestication-related genes from O. rufipogon and 20% from South/Southeast Asian wild O. nivara (Jing et al., 2023). Genes like An-1, BH4, PROG1, SH4, Rc, Rd, and GS3—which regulate awn length, hull color, tiller angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa (Yu et al., 2021). However, domestication and yield-focused breeding eliminated wild rice's valuable genes (e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.

Genome editing has boosted crop genetic improvement and wild plant de novo domestication—facilitating ideal crop development, food security, and sustainable low-input agriculture to expand human resources (Tang and Zhang, 2023). For example, via Oryza sativa functional genes, editing trait-controlling gene homologs rapidly improved agronomic traits in polyploid Oryza alta (CCDD genome; Yu et al., 2021). Although de novo domestication work based on polyploid wild rice has shown great value, considering the difficulty of directly domesticating polyploid wild rice from scratch, we propose using diploid wild rice as the starting material. By combining this with a multi-gene co-editing strategy targeting the An-1, BH4, PROG1, SH4, Rc, Rd, and GS3 genes, we aim to rapidly aggregate important agronomic traits in wild rice, provide alternative options for rice germplasm innovation, and offer experimental evidence for the role of these rice domestication or agronomic trait-related genes (Figure 1A).

See https://onlinelibrary.wiley.com/doi/10.1111/jipb.70087

Figure 1: Traits improvement of Oryza rufipogon W1681 and W1807 using multiplex genome editing systems.

(A) CRISPR-Cas9-based trait improvement workflow of wild rice. (B) W1807 transformation system (GFP fluorescence indicates successful transformation). (C) Collinear analysis of Oryza sativa Nipponbare (Nip), W1681, and W1807. (D) Number of resistant gene analogs in w1681, W1807, and Nip. NBS, nucleotide-binding site; RLP, receptor-like proteins; RLK, receptor-like kinases; TM-CC, transmembrane coiled-coil domain proteins. (E) Multiplex genome editing vector design and T₀ lines genotyping. (F) Differentially Expressed Genes (DEGs) analysis of WT versus quadruple mutants (−4m) spikelet transcriptome data. (G) Partial top KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment pathways. (H) Relative expression of DEGs. (I, J) Tiller angle comparison (I) and statistics (J) of WT versus septuple mutants (−7m, n ≥ 3). (K) and (N) Comparison of awn lengths, hull color, and seed length. (L, O) Grain and awn length statistics (n ≥ 3; n ≥ 18). (M) 1,000-grain weight (n ≥ 4). (P) Seed Breaking tensile strength (BTS) (n ≥ 16). (Q) Fracture surface of seed shattering (red arrow: smooth abscission layer). (R, S) Lignin staining (white arrow: complete abscission layer) (R) and quantification (S). Data: mean ± SEM; Student's t-test, **P < 0.01.