水稻穗粒数QTL qGN6 的图位克隆及功能分析

Grain number (GN) is the important component factor of rice yield. It is one of the most efficient way to increase rice yield through the improvement of grain number per panicle in high-yield rice breeding. A major quantitative trait locus (QTL) qGN6 of grain number was mapped on the long arm of chromosome 6 which drived from the segregating F2 population with parents of HR1128 and Nipponbare. The main QTL qGN6 was validated in the subsequent backcross segregation populations and it explained the highest phenotypic variation rate of 21.5%. The analysis of the near isogenic line (NIL) about the qGN6 indicated that the large panicle trait was controled by a dominant allele camed from the HR1128. The qGN6 allele can significantly increase the number of spikelets per panicle,the number of branches, panicle length and plant height, but has no obvious effect on the heading date, seed setting rate and grain weight.While the tiller number is significantly reduced, but the yield was significantly increased finally. Finally, the qGN6 was fine mapped into a 22Kb physical interval through the substitution mapping with the recombinant plants. Based on these findings, sequence comparison of this 22kb fragment between the dominant and recessive NIL-F2 individual at qGN6 locus, which will confirm the candidate genes; identification of the qGN6 gene from these candidate genes by complementary test. Moreover, protein subcellular localization, gene expression analysis, hormone determination and electron microscopy, transcriptome analysis, gene over-expression and deletion analysis and detection of allelic variations are worked to clarify the biological function of qGN6, and reveal its molecular mechanisms of regulating rice grain number development.

穗粒数是水稻产量性状的重要组成因子，通过改良穗粒数性状来提高产量是高产育种的有效途径之一。申请者前期在大穗品种HR1128与日本晴构建的F2群体检测到一个穗粒数主效QTLqGN6，并在后续回交分离群体中得到验证，qGN6最高解释了21.5%的表型变异；近等基因系分析表明，来自HR1128的等位基因表现为大穗显性，能够极显著增加穗粒数、穗分枝数，显著增加穗长、株高，对生育期、结实率和千粒重等无明显影响，虽然分蘖数是显著减少，但单株产量还是显著提高。进一步借助重组单株代换定位将qGN6精细定位至第6染色体22Kb范围内。项目拟在此基础上，开展对候选基因的测序及表达差异分析确定目标基因，通过互补试验验证目标基因。随后通过蛋白亚细胞定位、表达特征分析、激素测定和电镜观察、转录组学分析、基因过表达和缺失分析、等位变异检测，明确qGN6的生物学功能，初步揭示其参与水稻穗粒数形成的分子调控机制。

穗粒数是水稻产量性状的重要组成因子，通过改良穗粒数性状来提高产量是高产育种的有效途径之一。前期检测到一个主效的穗粒数QTL qGN6，来自大穗恢复系R1128的等位基因能够显著增加穗粒数、穗长和株高，定位在Chr6的22Kb区间内。在此基础上，本项目完成了基因克隆、功能验证等相关内容，构建了多套代换系、转基因材料，利用搜集的育种亲本开展了等位变异分析，通过转录组测序分析了功能基因表达情况，多途径探讨了qGN6的生物学功能。进一步将qGN6缩小至约13.98Kb，仅LOC_Os06g45460在近等基因系中差异显著，该基因也是已克隆的穗发育位点APO1；测序表明双亲差异包括2个碱基的替换（A-G; C-G）和1个9bp（CGGCGGCGG）缺失，均位于外显子中，与之前报道的CDS变异不同，可能是新的等位。遗传互补结果显示阳性株系的每穗粒数、穗长均显著高于对照，基因敲除株系也显现出小穗、株高降低等预期表型；qGN6在所检测的各个时期的组织中均有表达，在根和幼穗（1mm）中表达量最低，在5mm幼穗中表达量最高，定位于细胞核。不同长度片段互补出现表型差异，更长启动子互补片段能显著提升穗粒数（173.4 VS 117.7），但穗长没有增加，暗示与之前包含3Kb启动子互补片段存在不同的穗型调节效应。转录组显示包括OSMADS3、OsAAP5、qTGW3、OsSPL7、IPA1、TAW1等在内多个产量和花发育相关的基因出现差异表达；NIL-qGN6的IAA和GA3含量均要略高于NIL-qgn6。推测qGN6可能通过抑制相关下调基因的表达，同时上调某些位点（如IPA1）的表达，影响了相关激素的代谢，影响穗发育。基因聚合系显示qGN6和GN1a对水稻穗粒数具有累加效应，对穗长的增效作用也在新背景下得到更大的展现，而对株高的极显著提升需要进一步的试验来验证，这一结果为穗型及株型的聚合育种研究提供了信息参考。自然变异分析qGN6-CDS在1143份籼型材料中有14种单倍型，涉及其中的56份亲本；绝大部分材料的变异类型主要集中在Hap4、8和12。R1128与日本晴之间的9bp-INDEL差异很可能是籼粳间的分化差异，在育种群体中并未检测到，开发了对应的检测标记用于粳稻穗型的改良。