羽衣甘蓝花青苷积累关键基因精细定位与克隆

Kales (Brassica oleracea var. acephala) comprise various types with different leaf color and the red ones are rich in anthocyanin with high ornamental and nutritional value. As a kind of cruciferous plant with the most abundant variation in leaf color and anthocyanin content, kale can be a model to study leaf color variation and anthocyanin metabolic regulatory mechanism. Fine mapping and cloning the key gene controlling anthocyanin accumulation play crucial role in clarifying these scientific issues. In this project, two genetic segregation populations, an F1DH and an F2 population, which were constructed by two DH lines with significant difference of leaf color, will be used for fine mapping the key gene responsible for the anthocyanin accumulation in kale. We will construct a high-density SNP map of the F1DH population by SLAF-seq technology, as well as detect QTLs for anthocyanin content. QTL-seq analysis, which was based on genome-wide comparison of SNP profiles between red and white color “extreme bulks” constructed from F2 plants, will be conducted to identify major QTL of red leaf color. Combining QTL mapping and QTL-seq analysis, the key gene controlling anthocyanin accumulation will be mapped. Fine mapping will be done via further expanding F2 population with marker genotyping, as well as candidate gene will be identified by gene annotation information and sequence differential analysis. Then, the candidate gene will be cloned and differential expression analysis will be performed by qRT-PCR. The function of candidate gene will be certificated by transgenic analysis. This research will lay foundation of revealing the molecular genetic mechanism of leaf color and anthocyanin biosynthesis in Brassica oleracea, and of the breeding new cultivars with high anthocyanin content and unique qualities.

羽衣甘蓝叶色丰富，红色类型富含花青苷，具有很高的观赏和营养价值。作为十字花科作物中叶色与花青苷含量变异最丰富的种类，羽衣甘蓝是研究叶色变化规律和花青苷代谢调控机制的理想试材。定位并克隆花青苷积累关键基因是阐明这些科学问题的重要环节。本项目利用叶色性状稳定的红色和白色DH系构建的F1DH和F2遗传分离群体，通过SLAF-seq简化基因组测序构建高密度遗传连锁图谱，对羽衣甘蓝花青苷含量进行QTL定位；对F2群体构建红色和白色“极端池”，进行高通量全基因组重测序，根据红色亲本基因型在两个池中的SNP比率差异，对叶色性状进行QTL-seq分析。综合两种分析方法的定位结果，进一步对羽衣甘蓝叶色控制基因进行精细定位，确定候选基因，并对候选基因进行克隆测序、表达分析和转基因验证。本项目为阐明甘蓝类蔬菜叶片颜色和花青苷生物合成的调控机制，分子改良叶色和花青苷含量，培育高品质、有特色的新品种提供理论依据。

羽衣甘蓝叶色丰富，红色类型富含花青苷，具有很高的观赏和营养价值。作为十字花科作物中叶色与花青苷含量变异最丰富的种类，羽衣甘蓝是研究叶色变化规律和花青苷代谢调控机制的理想试材。定位并克隆花青苷积累关键基因是阐明这些科学问题的重要环节。本项目利用叶色性状稳定的红色和白色DH系构建的F1DH和F2遗传分离群体，通过基因组重测序构建高密度遗传连锁图谱，对羽衣甘蓝花青苷含量进行QTL定位；对F2群体构建红色和白色“极端池”，进行高通量全基因组重测序，根据红色亲本基因型在两个池中的SNP比率差异，对叶色性状进行QTL-seq分析。综合两种分析方法的定位结果，进一步对羽衣甘蓝叶色控制基因进行精细定位，确定候选基因。经过三年的项目研究，分析获得了羽衣甘蓝的花青苷在植株和叶片中的分布规律，并测定了花青苷的代谢谱；构建了包含1290个株系的 F2群体，建立了红的和白色叶片性状极端池，利用高通量测序QTL-seq 分析，通过位于 C06染色体上的选择信号定位到调控羽衣甘蓝叶片紫色的准候选关键调控基因 PAP1和 PAP2，并通过裂叶和圆叶极端性状池的测序和 QTL-seq 分析定位到了控制甘蓝裂叶的关键基因BoMLI1；利用小孢子培养技术构建了包含150个株系的 F1DH 遗传群体，通过对个株系的重测序分析，构建了包含1696个 Bin-marker 的高密度遗传连锁图谱，为进一步精细定位羽衣甘蓝重要农艺性状基因和分子育种奠定基础；利用转录组测序研究了红色和白色材料不同颜色叶片的表达差异，根据花青苷合成调控基因的差异表达建立了羽衣甘蓝叶片显色的遗传调控网络；为了更好地研究羽衣甘蓝重要农艺性状的遗传机制，利用三代和二代测序技术相结合的方法对羽衣甘蓝进行了全基因组重头测序。本项目研究取得的结果为进一步克隆羽衣甘蓝叶片颜色和形状等农艺性状的关键调控基因和研究基因的功能，以及分子设计育种奠定了理论基础；培育高品质、有特色的新品种提供理论依据。