白菜叶缘裂刻主效QTLlob10.1的克隆与鉴定

The lobed-leaf character is a clearly recognized morphological trait that has potential advantage for photosynthesis and resistance in plants. In our previous studies, the major QTL, lob10.1, controlling leaf lobes was identified in Brassica rapa depending on an F2 population derived from a cross between RcBr, with lobed leaves, and Chinese cabbage 08A061, with smooth leaf margin. We developed near isogenic lines (NIL), for the major QTL lob10.1 by using maker assisted background and foreground selection. And the constructed NIL were found to have 99.45% of the RcBr genome which was estimated by using 168 genome-wide markers for genotyping. Compared to the recurrent parent, RcBr, all the NILs showed the absence of leaf lobes and no significant differences in other agriculturally important traits. The genetic analysis showed that this trait is controlled by a single dominant gene in the RcBr genetic background. In the present study, the markers in the QTL confidence interval will be developed basing on the NIL and RcBr re-sequencing comparison and the major QTL lob10.1 will be fine mapped basing on NIL-F2 population which derived from the cross between RcBr and NIL. The candidate gene will be selected basing on their annotated functions, their differences of sequences and structures, gene allelic diversity and expression level of genes between NIL and RcBr. The function of the candidate gene will be validated by using overexpression and RNAi transformated to Arabidopsis and Brassica.napus. The expression characteristics of the candidate gene will be analyzed by tissue in situ-hybridization, promoter activity analysis and subcelluar location of gene products. Our results will shed light on deciphering the molecular mechanisms for leaf lobes formation and development regulation in B.rapa and it will also be useful for crop improvement of related species.

叶缘裂刻是易于识别的形态性状，也是与植物光合、抗逆相关的农艺性状。前期研究中在A10染色体末端定位一个叶缘裂刻主效QTL lob10.1，以叶缘深裂的RcBr为轮回亲本，分布于全基因组的168个标记前景背景辅助选择，构建了该主效QTL的近等基因系NIL。与轮回亲本比较，其背景恢复率99.45%，表现叶缘全缘，遗传分析表明，该性状在RcBr遗传背景下受单显性基因控制。本研究拟通过NIL与RcBr重测序开发SNP、InDel标记，基于NIL与RcBr构建的NIL-F2群体，精细定位lob10.1；通过基因功能注释分析、表达量差异、序列差异及多样性分析筛选候选基因；应用过表达和RNAi干扰转化拟南芥和甘蓝型油菜，验证候选基因功能，利用组织原位杂交、启动子融合表达和亚细胞定位技术，对其表达特性进行分析。研究结果有助于了解白菜类植物叶缘裂刻形成的分子机制，为相关作物叶缘性状的遗传改良提供参考。

叶片是植物的重要器官，其形态变化是适应环境条件的一种反映。叶缘裂刻不仅是一个易于识别的形态性状，也是一个与植物光合、抗逆等密切相关的重要农艺性性状，影响作物的商品性和产量。十字花科芸薹种包括了大白菜、芜菁和白菜型油菜等多个亚种，是重要的蔬菜和油料作物，芸薹种不同亚种、品种间叶片形态存在着广泛的变异。目前关于叶缘裂刻形成机制研究在芸薹种中，虽有一些报道，但其叶缘发育的分子机制还不清楚。项目前期研究中在A10染色体末端定位了一个叶缘裂刻主效QTL lob10.1，现以叶缘深裂的RcBr为轮回亲本，构建该主效QTL的近等基因系NIL及衍生的NIL-F2群体，开发SNP、InDel标记精细定位lob10.1；通过功能注释分析结合序列表达差异筛选候选基因；应用过表达和基因编辑技术转化RcBr，验证候选基因功能并对其表达特性进行分析。最终将lob10.1精细定位在A10染色体末端标记BraA10-26和BraA10-28之间，物理距离为69.8kb；候选区间内9个基因，RNA-seq显示只有BraA10g032440.3C（RCO）在两亲本之间差异表达,对RCO序列克隆分析表明，RCO基因全长1762bp，含有三个外显子和两个内含子，在RcBr的1004bp处出现了一个G碱基的插入，可能导致该基因移码突变，最终导致叶形突变。为了验证基因功能，对候选基因进行CRISPR/cas9敲除，结果使RcBr叶裂消失，且T1、T2代均表现为圆叶，表明该敲除可稳定遗传，进一步验证了RCO作为调控大白菜叶缘裂刻形成的候选基因。RCO转录因子作为HD-ZIPⅠ家族的一员，根据前人研究结果，结合DAP-seq和RNA-seq分析，寻找RCO 作用的下游基因，为进一步深入研究叶缘裂刻形成的分子机制奠定基础。研究结果有助于了解白菜类植物叶缘裂刻形成的分子机制，为相关作物叶缘性状的遗传改良提供参考。