Data_Sheet_3_In-Frame and Frame-Shift Editing of the Ehd1 Gene to Develop Japonica Rice With Prolonged Basic Vegetative Growth Periods.PDF

Japonica rice has become increasingly popular in China owing to its superior grain quality. Over the past decades, “indica to japonica” projects have been proposed to promote cultivation of japonica rice in low latitudes in China. Traditionally, japonica varieties were planted mainly in mid latitudes in the northeast plain and Yangtze River region. The key obstacle for introducing elite mid-latitude japonica varieties to low latitudes is the severe shortening of growth period of the japonica varieties due to their sensitivity to low-latitude short photoperiod and high temperature. Here we report development of new japonica rice with prolonged basic vegetative growth (BVG) periods for low latitudes by targeted editing the Early heading date 1 (Ehd1) gene. Using CRISPR/Cas9 system, we generated both frame-shift and/or in-frame deletion mutants in four japonica varieties, Nipponbare, Longdao16, Longdao24, and Xiushui134. When planting at low-latitude stations, the frame-shift homozygous lines exhibited significantly longer BVG periods compared with wild-types. Interestingly, we observed that minor deletion of the first few residues within the receiver domain could quantitatively impair the function of Ehd1 on activation of Hd3a and RFT1, resulting in an intermediate-long BVG period phenotype in the homozygous in-frame deletion ehd1 lines. Field investigation further showed that, both the in-frame and frame-shift lines exhibited significantly improved yield potential compared with wild-types. Our study demonstrates an effective approach to rapid breeding of elite japonica varieties with intermediate-long and long BVG periods for flexible cropping systems in diverse areas or under different seasons in southern China, and other low-latitude regions.

粳稻（japonica rice）因优异的稻米品质，在中国愈发受到欢迎。过去数十年来，中国提出了“籼改粳（indica to japonica）”项目，以推动粳稻在低纬度地区的种植推广。传统上，粳稻品种主要种植于中纬度地区的东北平原与长江流域。将优良中纬度粳稻品种引入低纬度地区的核心障碍，在于粳稻品种因对低纬度短日照与高温敏感，导致生育期大幅缩短。本研究通过靶向编辑抽穗期早1基因（Early heading date 1, Ehd1），培育出适配低纬度地区、具有延长基本营养生长期（basic vegetative growth, BVG）的新型粳稻。本研究借助CRISPR/Cas9系统，在日本晴（Nipponbare）、龙稻16、龙稻24以及秀水134这四个粳稻品种中，分别创制了移码突变与（或）框内缺失突变体。在低纬度试验站种植时，移码突变纯合株系的基本营养生长期较野生型显著延长。值得注意的是，本研究发现接收结构域内前少数氨基酸残基的微小缺失，可定量削弱Ehd1对Hd3a与RFT1的激活功能，进而使框内缺失ehd1纯合株系呈现出中等偏长的基本营养生长期表型。田间试验进一步表明，框内缺失与移码突变株系的产量潜力均较野生型显著提升。本研究提出了一种高效育种策略，可快速培育出具有中等偏长及长基本营养生长期的优良粳稻品种，以适配中国南方及其他低纬度地区不同区域或不同季别的灵活种植制度。