Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.

Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.

Transcriptional and post-transcriptional regulation of heading date in rice.

Rice is a facultative short day (SD) plant. In addition to serving as a model plant for molecular genetic studies of monocots, rice is a staple crop for about half of the world's population. Heading date is a critical agronomic trait, and many genes controlling heading date have been cloned over the last 2 decades. The mechanism of flowering in rice from recognition of day length by leaves to floral activation in the shoot apical meristem has been extensively studied. In this review, we summarise current progress on transcriptional and post-transcriptional regulation of heading date in rice, with emphasis on post-translational modifications of key regulators, including Heading date 1 (Hd1), Early heading date 1 (Ehd1), Grain number, plant height, and heading date7 (Ghd7). The contribution of heading date genes to heterosis and the expansion of rice cultivation areas from low-latitude to high-latitude regions are also discussed. To overcome the limitations of diverse genetic backgrounds used in heading date studies and to gain a clearer understanding of flowering in rice, we propose a systematic collection of genetic resources in a common genetic background. Strategies in breeding adapted cultivars by rational design are also discussed.