一个超级稻高产抗倒新位点的分子调控机制及其育种应用

The primary target in rice breeding is to develop varieties with high yield and lodging resistance, which is determined by different quantitative trait loci (QTLs). It showed that natural variations on non-coding region have important roles in traits determination, because those variations would pose impacts on mRNA transcription of nearby gene. It is significant to clarify the underlying mechanism of transcriptional regulation by the non-coding variations, which will facilitate the management of such loci. Our previous work identified a major QTL conferring the traits of strong culms and large panicle of Chinese super rice, and named it GSP1. Interestingly, no coding gene was found when the locus was dissected by map-based cloning. Expression analysis suggested that the locus exerts function by promoting expression of nearby gene, which was confirmed by later transgenic experiments. Therefore, the GSP1 locus covers a controlling region for gene transcription. In this project, the region will be over-expressed to identify its trans-acting activity and also subjected to GUS reporter analysis to clarify its cis-acting role. Moreover, DNA methylation, histone lysine modification, small RNA accumulation and chromatin structure in the GSP1 site will be analyzed to find the critical roles of different epigenetic modifications in shaping gene expression. After getting the underlying mechanism, we will perform both natural population analysis and CRISPR/Cas9 mediated genome editing to identify new GSP1 alleles with various promoting effects on traits performance，providing more selections for breeding application. Finally, we will clarify the combinative effect of pyramiding GSP1 module with qPL6 module, another QTL for high yield and lodging resistance from the same super rice. All of which will provide both theoretical and technical supports to fast develop varieties with high yield and lodging resistance.

高产抗倒伏是当前水稻育种的主要目标，相关性状主要由数量性状位点（QTL）调控，研究显示很多重要QTL的变异发生在非编码区，通过影响下游基因转录产生理想性状，研究不同变异类型改变基因表达的分子机制具有重要理论和应用价值。申请者前期工作定位了一个调控超级稻粗杆大穗性状的主效位点GSP1，该位点不包含任何编码序列，表达分析及遗传转化实验证明GSP1通过促进下游基因表达产生高产抗倒性状。本项目将进一步探索GSP1作用于下游基因表达的分子调控模式，明确不同表观遗传机制在改变基因转录过程中的作用；随后利用群体遗传及基因编辑技术筛选更多具有应用价值的等位变异，并探索GSP1分子模块与长穗抗倒伏模块qPL6促进超级稻优良性状形成的分子耦合模型，为快速提高水稻产量潜力及抗倒性提供理论基础与技术路线。

水稻重要农艺性状大都由数量性状变异位点（QTL）决定，是育种的重要筛选目标，目前水稻中克隆的许多重要位点都是通过改变基因表达产生理想性状，但前期很多研究主要针对蛋白功能开展研究，对相关变异如何决定基因表达研究较少。GSP1是一个调控粗杆大穗性状的QTL，是超级杂交稻高产的重要决定位点，精细定位显示其不包含蛋白编码序列，主要通过上调附近IPA1基因表达产生表型，根据等位基因命名法，我们也将其命名为ipa1-2D。序列解析发现该位点是一个三元重复序列，将该重复序列互补或超表达无法产生目标性状，证明其没有反式作用效应，报告基因分析显示其也不具备增强子功能，证明GSP1/ipa1-2D通过新的机制调控基因表达。甲基化分析显示IPA1基因处于超甲基化状态，对应转录抑制特征，而GSP1/ipa1-2D能够释放上述超甲基化状态，并产生开放的染色质结构，证明GSP1/ipa1-2D对基因的调控具有染色体位置依赖性。群体遗传分析发现GSP1/ipa1-2D是一个近期起源于中国的稀有位点，尚未广泛导入到世界各地的主栽品种中，对GSP1/ipa1-2D进行基因编辑可以创造更多的穗型变异类型，为育种提供了多重选择。与qPL6的聚合分析发现两者在穗二级分支存在竞争效应，一定程度上解释了无法通过聚合多个高产位点快速增产的育种现状，且GSP1/ipa1-2D的杂种优势表现依赖于qPL6基因型，为深入研究杂种优势理论提供了研究思路。