Mutations of Two Florigen Genes Have Different Effects on Controlling Flowering Time in Rice under Natural Long-Day Conditions

Flowering time of crops is critical for maximizing yield, quality and regional adaptability under target environments. In rice, Heading date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1) proteins are the key factors of flowering time regulation in the florigen activation complex. However, effects of hd3a and rft1 mutations on the flowering time under long-day conditions are not clear. In this study, we generated single and double mutants of Hd3a and RFT1 in japonica varieties Shen Nong 9816 (SN9816) background, termed hd3a-SNs, rft1-SNs and doub-SNs, by using CRISPR/Cas9 system. The flowering time of hd3a-SN mutants was similar to wild-type (WT); but mutations of RFT1 delayed flowering time by 11 to 14 days comparing to WT under nature long day (NLD) conditions. Double mutants of Hd3a and RFT1 (doub-SNs) exhibited an extremely late flowering phenotype, and they did not flower up to 365 days after regeneration under either NLD or short-day conditions. The leaf emergency rate of RFT1 mutants was similar to WT, suggesting that the late flowering phenotype of rft1-SNs was caused by delayed floral transition, but not by retarded growth. The expression of the key floral organ identity genes was significantly decreased in rft1-SN and doub-SN mutants, resulting in late flowering phenotypes. The expression profiles, evolution and haplotypes of Hd3a and RFT1 was also analyzed. Our studies provide insight into the potential utilization of Hd3a and RFT1 gene resources to improving growth period and regional adaptability in rice breeding.