水稻种子耐储藏主效基因qSC9-1的克隆和功能分析

Reduction of seed vigor during the storage of rice seeds, especially for hybrid rice, is a major ubiquitous problem in rice production. The fundamental way to improve seed storability in rice is to exploit and use storability-related genes in breeding practice. Though a number of QTLs associated with seed storability have been identified in primary mapping, none of them have been cloned yet. We previously identified a major-effect QTL qSC9-1 associated with seed storability in rice from the storage-tolerant indica line Guanghui 122. We have narrowed it down to an interval of 53.2 kb between the InDel marker D917 and SNP marker S904 on chromosome 9, using near isogenic lines (NILs) derived from residual heterozygous lines (RHLs), which were selected from the recombinant inbred lines (RILs) between the indica line Guanghui 122 and the japonica line Huajing 6.This project aims to clone and functionally characterize the qSC9-1 locus on the basis of our recent advancement. To this end, we will nominate the candidate gene after sequence comparison, and validate its biological function using genetic complementation, overexpression and RNAi approach. In addition, we will study the spatial-temporal expression pattern of qSC9-1 gene and the subcellular localization of its encoded protein. Moreover, we will analyze its allelic variations, geographic distribution, origin and domestication using a mini core collection of 110 accessions. Functional marker will be developed for marker assisted selection (MAS) as well. New findings from this study might provide scientific guidance for breeding storage-tolerant rice cultivars, and lay a solid foundation for elucidating the molecular mechanism underlying qSC9-1-mediated seed storability.

水稻种子尤其是杂交稻种子在储藏过程中活力下降，成为生产中的普遍问题。挖掘和利用水稻耐储藏基因，是改善水稻种子耐储藏性的根本途径。虽然有多个水稻耐储藏性QTL得到鉴定，但尚无相关QTL被克隆。我们选育的籼型恢复系广恢122具有良好的耐储藏性，前期研究从中发现控制种子耐储藏性的主效QTL qSC9-1。我们进一步构建广恢122/华粳6号的籼粳交群体，利用剩余杂合系衍生的近等基因系，将qSC9-1缩小到InDel标记D917和SNP标记S904之间的53.2kb范围。本项目在此基础上，拟对候选区间测序确定候选基因；通过遗传互补、超表达和RNAi等途径验证候选基因的功能；研究qSC9-1基因的时空表达模式和亚细胞定位；并利用110份核心种质分析qSC9-1基因的等位变异、地理分布、起源和驯化，开发功能标记。研究结果可为耐储藏水稻品种选育提供指导，并为阐明qSC9-1影响耐储藏性的分子机理奠定基础。

耐储藏性是水稻种子的重要农艺性状。水稻种子耐储藏性是多基因控制的数量性状。目前，已定位到多个控制水稻种子耐储藏性的QTL，但尚无相关QTL被克隆。项目组选育的籼型恢复系R122具有良好的耐储藏性，前期研究我们利用分离群体，从R122中鉴定到控制种子耐储藏性的主效QTL qSC9-1，位于第9染色体。本项目在精细定位的研究基础上，开展了亲本材料广恢122和HJ6的基因组重测序，完成对qSC9-1区间候选基因的测序和分析，开展了候选基因的组织表达分析，利用无缝克隆技术构建qSC9-1的遗传互补载体，利用CRISPR-Cas9技术构建qSC9-1的基因敲除载体，分别获得HJ6背景的遗传互补转基因植株和广恢122背景的基因敲除植株，对qSC9-1进行功能验证，分析了耐储藏基因SC9的保守结构域、基因共表达模式、同源进化关系及互作网络。同时，利用从Rice3K项目的SNP-Seek Database对3024份多样性水稻种质进行qSC9-1等位变异分析，挖掘其单倍型。进一步开发了功能性分子标记，通过杂交、回交和分子标记辅助选择，将qSC9-1位点导入具有华南生态区域特色的优质稻品种，获得了多个耐储藏性改良的品系。本研究为培育新型的耐储藏水稻品种奠定了基础，有助于阐明qSC9-1影响耐储藏性的分子机制。