整合传统遗传分析和新一代测序数据以解析数量性状多基因分子遗传机制的理论和实验研究

Most traits are polygenically controlled and environmentally modified, including those threatening human health, controlling reproductive isolation between species and also those agronomically important in breeding for high yield, better quality and improved adaption of animals, plants and microbes. Understanding the molecular mechanisms underlying polygenic variation has been one of the most challenging areas in both history of genetics and the era of modern function genomics. The genome-wide marker assisted mapping of quantitative trait loci (QTL) has greatly widened the window of quantitative genetic analysis. However, it is still a task with an exceptionally low success to dissect QTL to a genic level due to the bottleneck of current linkage and linkage disequilibrium based QTL mapping strategies in both mapping precision and resolution. To address this fundamental and challenging question, the proposal is designed to develop novel theoretical and experimental strategies to unveil the molecular basis underpiining quantitative genetic variation at genic, transcriptional and their interactional levels. To achieve this, we propose to develop novel theoretical frameworks and analytical tools that enable integration of genome and transcriptome sequence data from the populations created from recurrent bi-directional selection and backcrossing (RSB) breeding schemes. Feasibility, reliability and utility of the theoretical analyses and experimental strategies will be tested by experimentally exploring ethanol tolerance of budding yeast as the experimental model of quantitative traits. This project aims to open a new route for understanding molecular basis of complex genetic architecture of quantitative traits for the species to which the breeding activities such as backcrossing and phenotypic selection are ethnically possible.

大多数影响人类健康、物种生殖隔离、动植物及微生物育种的重要经济性状（例如产量、品质、抗逆性以及环境适应性）均属于受多基因遗传控制和环境因素共同决定的数量性状。认识并解析数量性状复杂的多基因分子遗传结构和功能机制是功能基因组学最具挑战性的研究领域之一。目前，借助基因组中广泛分布的DNA遗传标记辅助QTL定位研究，为在基因组水平探索和认识数量遗传变异的遗传机制取得了重大进展。但是，在基因的分子结构和功能水平解析数量性状变异分子机制仍限于瓶颈。本项目建议发展整合传统遗传分析和多重新一代组学测序数据，以在基因组的结构和表达调控功能的多层次，解析数量性状遗传变异的分子机制的理论和实验研究策略。以酿酒酵母乙醇耐受性为数量性状的实验模型，实际验证该理论和实验策略的可行性、可靠性以及局限性。本项目研究的开展和成功完成将为复杂数量遗传变异分子机制的研究开辟一条新的方向，并为相关研究提供有效分析工具。

本项目以酿酒酵母为实验模型，以酿酒酵母细胞有丝分裂子母细胞分离变异，这一重要的数量性状为研究对象，我们首先建立了简易、可靠的测试菌株有丝分裂子母细胞分离数量性状表型的实验方法；通过双向定向选择获得了有丝分裂子母细胞分离表型呈显著的亲本菌株，并以该亲本菌株构建了用于定位该数量性状主效基因的定位分离群体。；通过对基因组遗传标记辅助高精度、高解析定位，我们定位了控制该性状的三个主效基因的QTL。这三个主效QTL等位基因在分离群体的可以解释45%的表型变异，其中定位在酿酒酵母第II号染色体的QTL，其相邻遗传标记等位基因在作图群体中的分离能解释性状表型变异的25%。进一步的精细定位，将该QTL进一步缩小到包括仅包括5个候选基因的区域。通过逐一基因剔除(knockdown)实验，初步确定QTL候选基因Amn1。利用上述亲本菌株间Amn1等位基因互换实验以及在广泛的酿酒酵母菌株的Amn1等位置换实验，进一步验证Amn1即是控制酿酒酵母有丝分裂子母细胞分离数量性状表型的主效QTL基因。通过构建一系列Amn1的突变体，澄清了导致其表型变异的功能序列。由此，我们成功实现了本项目拟定的从QTL的精确、高解析定位到QTL基因分离分离的目标。