甘蓝型油菜每角果粒数主效QTL（qSS.C9）的图位克隆及功能分析

Seeds per silique (SS) is one of the most important components for seed yield in oilseed rapa (Brassica napus). However, the genes for this trait and their underlying molecular mechanism remains unclear. Based on near isogenic lines (NILs) for a major quantitative trait locus (QTL) qSS.C9 of the Brassica napus C9 chromosome, our previous study has delimited it to a physical region of 265 kb in Brassica olereace C9 chromosome. Recently,we further fished the Brassica napus BAC clones covering this region and obtained partial sequence information of them. Gene prediction showed that one ORF homologous to an Arabidopsis gene, is most likely the candidate of qSS.C9. In this project, we would preferrentially test this candidate gene, and continue to narrow the candidate region by using a large F2 population derived from the NILs. The candidate gene will be confirmed by functional complementation analysis. In combination with the data from cytological observation, haplotype analysis, temprol and spational expression, subcelluar localizaion of GFP-fused protein and overexpression as well as RNAi analysis, we would provide the insights of the biological function of qSS.C9, and screen the elite alleles of this locus. The downstream genes regulated by qSS.C9 would be identified through transcriptome sequencing, and further investigated with the aid of Arabidopsis genes related to the seed formation. Combining all these data, we would propose the molecular model about how the qSS.C9 locus controls the variation of seed number in siliques. Results from this research would lay a foundation for the better understanding of the molecualr mechanism of SS in Brassica napus, and provide elite allele resources as well as their functional markers for the true breeding programs.

每角果粒数是油菜产量构成的重要因子之一，但其分子遗传机制仍然不清。前期利用C9染色体每角粒数主效QTL qSS.C9的近等基因系，已将该位点锁定在甘蓝C9染色体265 kb的区段内，并已获得覆盖该区段的甘蓝型油菜BAC克隆及其部分序列。候选基因分析发现其中一个拟南芥同源基因极可能是qSS.C9的目标基因。本项目将优先验证该候选基因，同时利用近等基因系群体缩小候选基因区段至30 kb之内，最终通过互补分析获得目标基因。结合细胞学观察、单倍型分析、时空表达、蛋白亚细胞定位及增强和抑制表达的表型，明晰qSS.C9的生物学功能，筛选优异等位基因型并开发其功能标记。鉴定qSS.C9的下游基因及可能的互作，结合拟南芥种子形成相关基因的功能，提出qSS.C9调控种子数目变异的分子机理。该研究将为从分子水平解析油菜每角果粒数的变异奠定基础，并为该性状的遗传改良提供优良等位基因及其功能标记。

本项目在前期精细定位每角果粒数的主效QTL位点qSS.C9的基础上，采用图位克隆和比较基因组学相结合的方法成功分离了qSS.C9的目标基因。该基因编码一个与拟南芥AtSMG7（At5g19400）同源的蛋白，命名为BnaC9.SMG7b。序列分析表明，该基因在近等基因系之间存在PAV类型的多态性，在籽粒数目少的材料中，该基因完全缺失；虽然BnaC9.SMG7b在DNA和cDNA水平上和SMG7之间相似性极高，但其最终却只编码一个包含113个氨基酸的小蛋白。分子鉴定表明BnaC9.SMG7b的表达具备明显的时空表达特征，而BnaC9.SMG7b蛋白定位在细胞器P小体内，和拟南芥SMG7蛋白一致，这一表达模式的保守性和变化，表明其功能和拟南芥中同源基因之间存在一定的分化。细胞学研究发现，少粒材料NIL(HZ396)中65.6%的大孢子母细胞未形成正常的功能大孢子，但减数分裂到四分体过程正常，这一比例与NIL(HZ396)最终的胚珠败育率（69.8%）相似。结合拟南芥同源基因SMG7参与第二次减数分裂过程的步出调控，我们的研究结果表明BnaC9.SMG7b在大孢子母细胞减数分裂过程中的作用与拟南芥同源基因SMG7的功能一致，但其并不参与小孢子母细胞减数分裂过程的调控。通过对RNA-seq数据分析、油菜单拷贝基因+/-PTC转录本的表达量分析及遗传转化实验分析，表明BnaC9.SMG7b不参与无义突变介导的mRNA降解途径（NMD）。在自然群体中，BnaC9.SMG7b存在四种单倍型，单倍型 I 中 BnaC9.SMG7b 基因缺失，单倍型 II、III 和 IV 中的 SNP位点不改变氨基酸的编码。单倍型 I 与单倍型 II、III 和 IV 的每角粒数之间均存在显著性差异，而单倍型 II、III 和 IV 之间每角粒数不存在显著性差异，暗示着BnaC9.SMG7b 位点经历了明显的人工选择过程。综上所述，本项目组在油菜中第一次克隆到控制每角果粒数的主效QTL位点 qSS.C9，该基因编码一个与SMG7同源的小蛋白BnaC9.SMG7b，通过调控大孢子母细胞减数分裂的步出从而控制雌配子体的发育，最终调控每角果粒数。