水稻产量性状杂种优势新主效QTL的克隆和功能研究

Wide application of heterosis has played an important role in improvement of rice grain production since 1960s. However, its molecular basis needs to be extensively investigated. Yield of hybrid rice Liang–You–Pei–Jiu (LYP9) was found largely attributed to over-dominance in grains per panicle. One major quantitative trait locus (QTL) associated with heterosis for grains per panicle, qSN-p8, was thereby fine mapped within 160 kb on chromosome 8 using F2 population derived from a residual heterozygous line (RHL) chosen from F9 developed by 93-11 and PA64s. In the project, with developed single nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers, the novel QTL is to be isolated via map-based cloning strategy using a BC5F2 population by backcrossing one of LYP9 recombinant inbred lines (RILs) with PA64s qSN-p8 segment to 93-11. Complementation test, over-expression analysis and the clustered regularly interspaced short palindromic repeat-associated endonuclease 9 (CRISPR/Cas9) system will be applied in functional confirmation of candidate genes for qSN-p8. Comparisons are also to be conducted between a near isogenic line (NIL) NIL-qSN-p8 and NIL-qSN-p8-F1 produced by backcrossing the NIL to 93-11, a complementation line T1-qSN-p8 and T1-qSN-p8-F1 produced by crossing the line to the transformed parent, respectively at phenotypic, cellular and transcriptomic level. The study of regulatory network of genes based on single heterotic locus will contribute to both exploration of the genetic mechanism for heterosis concerning rice yield related traits and further exploitation of heterosis.

杂种优势的利用为我国乃至世界的水稻产量作出了巨大贡献，但是对其分子遗传基础的认识还有待深入。杂交稻两优培九在穗粒数方面具有正向超亲优势，是影响杂种F1产量的主要因素。因此，我们利用培矮64s和93-11的F9群体选出的剩余杂合体自交得到F2群体，已将新的控制穗粒数超亲优势主效QTL——qSN-p8精细定位在水稻第8号染色体的160 kb范围内。在此基础上，本项目利用两优培九重组自交系与93-11回交的BC5F2群体，发展SNP和InDel标记，对这一至今未见报道的杂种优势QTL进行图位克隆，并通过互补实验、过表达和CRISPR/Cas9技术验证基因功能；同时，对此QTL的近等基因系及其与93-11回交的F1、对互补纯系及其与转化亲本回交的F1在表型、组织细胞学和转录组水平上加以比较。在单位点基础上从基因网络角度来探究水稻产量相关性状杂种优势的遗传机制，将对杂种优势的进一步利用起指导作用。

杂种优势的利用为我国乃至世界的水稻产量作出了巨大贡献，作为水稻单产的三大要素之一的穗粒数是水稻杂种优势的重要体现。本项目利用超级杂交稻两优培九重组自交系与93-11回交的BC5F2群体，发展SNP和InDel标记，精细定位和图位克隆了一控制穗粒数杂种优势主效QTL——qSN-p8。双亲在SN8基因的启动子区2kb内存在2个SNP、外显子内存在引起氨基酸改变的2个SNP，并且培矮64s的SN8基因在孕穗早期幼穗中的转录水平表达显著高于93-11。基因敲除实验验证了SN8基因的调控二次枝梗数和穗粒数的功能。GUS染色发现SN8蛋白在根、叶和穗中表达，亚细胞定位将其定位在细胞核和细胞质中。SN8近等基因系与93-11回交得到的F1，其二次枝梗数和穗粒数与近等系和93-11差异显著且高于中亲值，证实SN8基因为控制穗粒数杂种优势的主效QTL。93-11、近等系和F1幼穗中基因转录水平表达比较显示：水稻二次枝梗数和穗粒数与SN8基因的表达量相关，并且DEP1基因在三者的表达模式与SN8基因一致。通过对培矮64s、93-11和两优培九孕穗早期幼穗的转录组测序，两两间比较分析发现了475个共有的差异表达基因，miRNA测序显示有227个miRNA的转录表达在培矮64s和93-11的穗中存在显著差异。利用SN8基因与已知其它穗分支基因聚合，成功育成二次枝梗数和穗粒数显著增加的高产优质杂交籼稻“深两优7248”。该水稻产量杂种优势主效QTL的克隆和功能研究，不仅初步揭示了水稻穗粒数杂种优势的遗传机制，为水稻杂种优势相关系列基因的挖掘提供了线索，而且作为宝贵的基因资源应用于超级稻育种，对杂种优势进一步利用起指导作用。