基于渗入系精细定位与克隆长药野生稻高产大穗基因Gn2

In the last decades, great achievement have been made in the rice genetic improvement and germplasm innovation. A case in point is that China produce over 30% of the global rice production via about 20% of the rice growing area in the world. Accompanying with this is the loss of genetic diversity and latent valuable genes in the modern rice varieties because of the strong human selection, which thus results in the almost stagnant increase for the grain yield improvement. Wild relatives of rice which live in the natural environment contain a lot of valuable mutations including high grain yield genes and QTLs so as to adapt itself to various stresses. As the rapid development of modern biotechnology, a lot of grain yield-related QTLs have been identified in the common wild rice in the latest years. However, there is no any other reports involved in the characterization and cloning of genes or QTLs for high grain yield from other wild rice species. O. longistaminata, a wild relative of rice in the AA genome, which is characterized with high and sturdy stature, long tassel and anther, show strong potentiality for rice improvement. To this end, we constructed a stable BC2F8 inbred backcross population derived from 9311 X O. longistaminata, and observed 24 important agricultural traits. Fortunately, the line IL1880 showed great many more of spikelets and higher grain yield than the receptor parent 9311. Which was characterized to be controlled by a single semi-dominant gene Gn2 on the chromosome 4. Further, we developed a near isogenic line from IL1880 and hence constructed a F2 population for cloning of the Gn2 gene. Then, using an array of molecular and cytological approaches to perform the research about the genetic mechanism of Gn2 how to regulate the grain yield. This research will provide us an effective manner to improve rice yield, and novel insights into the origin and evolution of genes related to grain yield.

过去半个世纪以来，我国水稻种质创新取得了突出成就。但强烈的人工选择，导致现代水稻品种遗传多样性不断窄化；水稻单产增长缓慢，表现出强烈的遗传瓶颈。野生稻长期处于自然状态，积累了多种优良变异，包括高产基因。随着现代生物技术的发展，普通野生稻高产基因的发掘取得了巨大进展，但其他野生稻种高产基因的发掘利用尚未见报道。长药野生稻具有株型高大，茎秆粗壮，长穗、大花药等特性。为发掘利用其有利基因，我们以9311为受体构建了一套含148个株系的 BC2F8稳定渗入系，通过24个重要农艺性状的考察，发现株系IL1880茎秆粗壮，穗大粒多，产量明显高于回交亲本9311，遗传分析显示其含有一大穗高产基因Gn2，初步定位在第4染色体。在此基础上，构建了以9311为背景的近等基因系。进而以近等基因系构建F2大群体,精细定位、克隆该基因，研究其遗传效应和分子机理，为水稻高产改良提供新的基因资源。

为发掘利用野生稻有利基因，我们采用图位克隆法克隆了长雄蕊野生稻控制一次枝梗的大穗基因Gn2，并初步分析了其作用机理。同时，通过比较杂交稻红莲优6与其双亲9311和粤泰A，克隆鉴定了一个调控水稻二次枝梗和穗粒数的关键基因miR396b，并阐明了其作用机理。.（1）我们以9311/L1880的F2构建了一个8000株的定位群体，参照日本晴基因组，将L880控制一次枝梗的基因Gn2（Gn8.1）定位在第8染色体长臂一个71.1Kb的区段。比较长雄蕊、日本晴和明恢63在该区段的编码基因发现，日本晴、L880和明恢63除均含6个相同的蛋白编码基因外，L880还含有一个栽培稻所没有的新基因，与大穗共分离且在穗中高表达，推测为Gn2。同时在Gn2位点，9311与L1880都含有大穗基因OsSPL14，可彼此仅启动子区有一SNP差异，但OsSPL14在L1880中的表达比9311高3.5倍，推测这种差异可能来自表观修饰。染色质组蛋白修饰分析发现，9311的H3K27me2 及H3K27me3修饰程度显著高于L1880，且9311中OsSPL14启动子-900至-600bp区间内胞嘧啶的甲基化水平也明显高于L1880；说明GN2主要通过H3K27me2与H3K27me3修饰来调控OsSPL14的表达，从而影响水稻的一级枝梗数。.（2）为了分析Gn2在杂交稻中利用的可行性，我们以L1880分别与蜀恢527、珞扬6号和9号杂交，聚合大穗基因Gn2、恢复基因Rf3~Rf6、抗稻飞虱基因Bph6和Bph9，借助分子标记辅助选择，与9311回交2代后，在118个BC2F1株系中选育出52个聚合了Rf3-Rf6、Gn2、Bph6及Bph9的七基因株系。抗虫、测产和恢复性鉴定表明，这些株系不仅产量高、抗稻飞虱，配合力好，而且恢复红莲、野败和两系不育系，具有良好的应用前景。.（3）通过比较杂交稻红莲优6及其亲本9311和粤泰A幼穗中miRNA表达，发现17个存在显著差异，通过遗传互补验证分析表明miR396b是调控二级枝梗伸长和颖花分化的关键基因。进而通过遗传和分子分析发现其功能是通过下游的靶基因GRF6来实现的。而GRF6属于转录因子，它直接上调幼穗中调控生长素合成与信号传导相关基因ARF7和ARF11、以及调控枝梗分化的MADS34和小花分化的TAWAWA1基因表达，从而促进穗子变大，提高产量。