控制葡萄果肉着色的QTL定位、候选基因挖掘和调控机制解析

Anthocyanin is one of the most important phytochemicals which, to a large extent, determines market price of grape berry and relative products. Anthocyanins are usually found in the skin of red grapes, but also in some special grape cultivars whose flesh is red-colored. While genetic mechanisms controlling synthesis of anthocyanins in berry skin have been well understood, anthocyanin production in red flesh is very limited. Elucidation of the genetic mechanism of anthocyanin synthesis in grape red flesh will help fill a critical knowledge gap in anthocyainin synthesis in grapes and accelerate marker-assisted grape breeding in the future. In this proposed project, we will firstly use HPLC to study the genetic variation and inheritance of anthocyanin content in red flesh of berry samples from a Vitis F1 population, which was created by crossing a white flesh maternal parent and a red flesh paternal parent, and some representative red flesh grape cultivars; Secondly, we will develop single nucleotide polymorphisms (SNP) using next generation sequencing (Genotype by Sequencing), and construct a high-density genetic map for the population, and then identify QTLs for genetic control of anthocyanin synthesis and content. Thirdly, we will obtain RNA-seq profiles of some representative offspring individuals from the mapping population and associate candidate genes identified from the RNA-seq profiles with the main QTLs identified from the QTL mapping. Finally, we will clone full length of most promising candidate genes and determine their roles in controlling anthocyanin synthesis and content in grape flesh. The function of candidate genes will be confirmed using a grape cell suspension transformation system.

花色苷是影响葡萄果实品质及相关产品市场价值的重要因素之一。在一些葡萄品种中，果肉中含有大量花色苷，这些花色苷合成的遗传机制尚不清楚。研究红肉葡萄中花色苷合成的遗传机制，对指导葡萄分子育种，加速葡萄新品种的选育有重要意义。本课题拟选用白肉母本和红肉父本杂交获得的葡萄F1代分离群体和部分红肉品种为材料，首先，利用果树生理学和遗传学方法，研究花色苷在果肉中的表型变异和遗传分离规律；其次，利用新一代测序技术结合简化基因组测序技术开发高通量SNP分子标记，构建高密度遗传图谱，通过花色苷有无和含量多少的表型数据与遗传图谱提供的基因型数据结合，扫描获得控制葡萄果肉中花色苷有无和含量的QTL；再次，利用极端个体转录组差异表达结果，结合生物信息学技术手段，挖掘控制果肉中花色苷合成的关键候选基因和连锁分子标记；最后，通过克隆并分析候选基因的全长序列，寻找其调控机制，并通过细胞悬浮转化系统验证候选基因功能。

花色苷是影响葡萄果实品质及相关产品市场价值的重要因素之一。在一些葡萄品种中，果肉中含有大量花色苷，这些花色苷合成的遗传机制尚不清楚。研究红肉葡萄中花色苷合成的遗传机制，对指导葡萄分子育种，加速葡萄新品种的选育有重要意义。本课题选用本研究以蘡欧杂种葡萄‘北丰’（V. thunbergii×V. vinifera）（果皮红色、果肉白色）与欧亚种染色品种‘烟73’（V. vinifera）杂交群体，通过简化基因组测序共开发1409个SNP标记用于构建杂交父母本遗传图谱，结合杂交群后代花色苷含量，鉴定了一个控制葡萄果肉找色的位于2号染色体的QTL。结合QTL定位和转录组差异表达数据，VvMYBA2基因不仅位于果肉着色的QTL位点内，同时发现VvMYBA2基因也是红色果肉与白色果肉葡萄转录组的差异表达基因。对多个品种VvMYBA2启动子序列进行序列比对分析，结果发现在红色果肉葡萄品种中存在8个SNP位点。荧光素酶结果显示VvMYBA2rf的活性显著高于VvMYBA2wf。将VvMYBA2rf启动子融合VvMYBA2基因，形成由VvMYBA2rf启动子控制的瞬时表达载体。转化农杆菌后瞬时转化烟草叶片，结果显示VvMYBA2rf可以诱导VvMYBA2基因产生花色苷，导致烟草叶片着色。研究结果为未来红肉葡萄育种提供了有效的分子标记，将有助于提升育种效率。