A higher-yield hybrid rice is achieved by assimilating a dominant heterotic gene in inbred parental lines.

The exploitation of heterosis to integrate parental advantages is one of the fastest and most efficient ways of rice breeding. The genomic architecture of heterosis suggests that the grain yield is strongly correlated with the accumulation of numerous rare superior alleles with positive dominance. However, the improvements in yield of hybrid rice have shown a slowdown or even plateaued due to the limited availability of complementary superior alleles. In this study, we achieved a considerable increase in grain yield of restorer lines by inducing an alternative splicing event in a heterosis gene OsMADS1 through CRISPR-Cas9, which accounted for approximately 34.1%-47.5% of yield advantage over their corresponding inbred rice cultivars. To achieve a higher yield in hybrid rice, we crossed the gene-edited restorer parents harbouring OsMADS1 with the sterile lines to develop new rice hybrids. In two-line hybrid rice Guang-liang-you 676 (GLY676), the yield of modified hybrids carrying the homozygous heterosis gene OsMADS1 significantly exceeded that of the original hybrids with heterozygous OsMADS1. Similarly, the gene-modified F hybrids with heterozygous OsMADS1 increased grain yield by over 3.4% compared to the three-line hybrid rice Quan-you-si-miao (QYSM) with the homozygous genotype of OsMADS1. Our study highlighted the great potential in increasing the grain yield of hybrid rice by pyramiding a single heterosis gene via CRISPR-Cas9. Furthermore, these results demonstrated that the incomplete dominance of heterosis genes played a major role in yield-related heterosis and provided a promising strategy for breeding higher-yielding rice varieties above what is currently achievable.