水稻粒形与穗着粒密度基因GDS7的克隆与功能分析

Grain weight (GW)and spikelets per panicle (SPP) are two important components of grain yield. GW is determined by grain size (GS). Negative correaltion between GW and SPP is observed and is frequently hard to balance the two traits in the rice breeding program beacuse the mechanism underlying the nagtive correlation is still unclear. In our previous work, we mapped a QTL for GW and a QTL for SPP in the same region on chromosome 7 using a recombinant inbred line population derived from the cross between Nanyangzhan and Chuan7. Progeny test of a near isogenic F2 population confirmed that it was a gene (named GDS7) simultaneously regulates both GW and SPP. Subsequently GDS7 was limited to a 55-kb region using a larger near isogenic F2 population with 6 recombinants left between flanking markers. Based on these results, we desgin the research project. GDS7 will be narrowed to a 15-kb region by developing new polymorphic markers in the target region. Further, 1-2 candidates of GDS7 will be chosen according to gene annotations, parental comparative sequencing and the expression patterns. The constructs contained candidate genes will be transformatted to callus and trangenic plants will be investigated for genetic complementary test. Finally we definitely claim GDS7 will have been successfully cloned. We will isolate the full length cDNA by RACE technique and clearly claim the structure of GDS7. qRT-PCR and in situ hybridization will be performed to examine the temporal and spatial expression patterns for the two homozygotes of near isogenic lines. Furthermore, we will deep sequence GDS7 for Chinese rice core collection and wild rice. Association analysis between the nucleotide polymorphism sites and the two phenotypes will make for searching favorable alleles for rice breeding. We will compare the nucleotide diversity between cultivars and wild rice, and try to determine whether GDS7 suffered selection and what type selection it suffered. Cloning of GDS7 provides us a good chance to elucidate the mechanism underlying the negative correlation between GW and SPP.

粒重与每穗颖花数是水稻产量性状的主要构成因子。粒重由粒形决定，并与每穗颖花数呈负相关，由于这种负相关的分子机制不明，这种负相关很难在育种中克服。我们在南洋占／川7组合衍生的重组自交系群体发现一个同时控制粒重与每穗颖花数的QTL区域，随后证实是一个单基因控制这两个性状，并把它定位在55 kb内，该基因命名为GDS7。在此基础上，本研究拟将GDS7精细定位至15-kb，并根据基因预测信息、亲本比较测序以及预测基因表达模式来确定1-2个候选基因。通过功能互补试验，确认克隆了GDS7。利用RACE技术分离它的全长cDNA，确认GDS7编码模式。利用qRT-PCR与原位杂交技术分析它的时空表达特异性。此外，对自然群体和野生稻进行GDS7深度测序和关联分析，鉴定优良等位基因，明确它在进化过程中是否受到选择，受到何种选择。GDS7的克隆对阐述粒重与每穗颖花数负相关调控机理提供条件。

本项目以水稻材料“南洋占”与“川 7” 衍生的6890个GDS7 （穗型与粒形基因）近等基因系F2株系，将GDS7精细定位至20-kb区间，并确定了2个候选基因（ORF1与ORF2）；通过遗传转化功能互补验证得出：ORF1是GDS7，进而证实该基因被克隆；研究发现GDS7的启动子区或者调控区的DNA变异是引起表型变异的原因。Real-Time分析得出：GDS7 两等位基因（NN与CC）在幼穗3期中存在表达差异性；进而证明GDS7的表达差异导致了NIL中的表型变异。对本室水稻团队529份水稻种质群体进行穗型表型收集，并结合已有的SNP数据对二次枝梗数(NSB)、每穗颖花数(SPP)与复合性状NSB/NPB进行了GWAS分析，其结果表明：复合性状NSB/NPB被检测到与GDS7存在显著关联；GDS7单倍型分析找出了利于增加NSB/NPB与SPP的单倍型。对GDS7近等基因系两基因型的颖壳分别纵切进行细胞形态观察与分析，同时结合RNA-Seq数据得出：GDS7参与调控细胞周期相关基因的表达，进而调控细胞分裂引起粒长变异。