基于GWAS分析和QTL定位研究枳落叶性状调控的遗传规律和分子机理

“Citrus was changed into trifoliate orange when it moved to northern part of China” has been legend for thousands of years. However, few persons pay attention to the evolution mechanism and potential scientific value behind this legend. Leaf dropping in citrus is usually considered as a bad and abnormal phenomenon under environmental stress such as cold, drought and waterlogging etc. In contrast, leaf dropping ahead of winter is a survival ability for trifoliate orange to enhance its resistance to cold temperature and thus allow it spreading from subtropical area to temperate zone in northern part of China. This project aims to reveal the inheritance and controlling mechanism of deciduous character in trifoliate orange. In order to overcome the interference of nucellar embryo, the interspecific hybridization is designated by using a precocious trifoliate orange with dominant morphology marker and GFP marker as male parent to cross with Citrus clemantina Hort. and Carrizo citrange. The inheritance of genes controlling the evergreen/deciduous trait will be analyzed according to the separation ratio among the constructed segregant population. Based on the BSA-seq analysis in the past year, an additional Genome-wide Association Analysis to the BC1 population will be carried out to construct a fine linkage map and thus to predict the precise gene locus controlling deciduous trait in trifoliate orange. The candidate genes will be screened by gene structure comparison based on genome re-sequencing, as well as co-separation analysis and the consistence of expression pattern with leaf dropping. At last, the function of object genes will be confirmed by gene transformation. The success of this project will help to understand how the trifoliate orange evolved the ability of sensing the seasonal climate change and raising its resistant ability, and thus provides a novel strategy for cold resistance breeding as well as applicable molecular markers and valuable genetic resources.

“橘逾淮为枳”已流传千年，但其背后隐藏的科学价值却少有人关注。 橘向枳变异的一个显著标志是落叶，落叶在柑橘中常被当作冻、旱、水涝等逆境下的不良表现，却是枳进化获得的保命本领：枳冬前落叶休眠后抗寒性增强，生存区从温暖的亚热带扩展到寒冷的温带。本项目研究旨在揭示枳落叶性状遗传规律、定位相关调控基因。为克服珠心胚干扰，拟采用带显性形态标记和GFP荧光标记的早实枳作父本，分别与克里曼丁橘和卡里佐枳橙杂交，构建可靠的杂交分离群体，根据常绿/落叶表型分离比例分析落叶性状遗传规律; 在前期BSA-seq连锁分析基础上，进一步采用回交后代分离群体的全基因组关联分析（GWAS）法，构建高密度连锁遗传图，并对落叶调控的相关基因进行精细QTL定位。通过基因结构比较、共分离检测、表达与表型一致性分析​筛选候选基因，最后以转基因验证基因功能。研究结果有助于解析枳落叶抗寒分子机理，为柑橘抗寒育种提供新思路和新材料。

落叶性状的发生是植物进化过程中的一个重要里程碑，是影响物种分化、进而扩大地理分布的重要事件。柑橘为世界第一大果树，是典型的亚热带植物。冬季极端低温是决定柑橘存活和栽培区域的限制因子。因此探明枳的落叶性状与其极强的抗寒性有无内存联系，了解落叶性状的遗传规律，解析枳落叶调控的分子机理具有重要的理论意义和潜在的应用前景。本研究以具有落叶性状并携带三出复叶显性标记的枳为父本，以单胚性状的克里曼丁橘或柚为母本，分别构建了F1代、F2代及BC1代群体，通过多个组合后代落叶、半落叶及常绿性状分离比例分析，判断枳落叶性状遗传符合双基因受2个基因共同作用，即由d (deciduous)和感受低温与干旱的信号因子 r （responding to environment factor）控制，dd为落叶的执行者，r或R为dd开关的决定者， R基因抑制d基因的执行，对d基因有上位效应，推测柚的基因型为RRDD，而早实枳的基因型为rrdd，而克里曼丁橘的基因型为RrDd。在枳柚杂交F2代中随机挑选出落叶、常绿各10株，在室温（24℃）、-4℃、-8℃和-12℃下分别处理6小时，之后以电导率法测量各处理枝条的细胞质膜伤害情况，结果表明，落叶植株的抗寒性强于常绿植株，说明落叶与抗寒之间是相关的。对落叶前后枳与克里曼丁橘叶片及离层部位的脱落酸含量、乙烯释放速率测定表明，枳离层部位的乙烯含量要高于常绿柑橘，可能有利于形成离层，导致叶片脱落。对落叶前后枳与克里曼丁橘枝条MDA、可溶性蛋白、可溶性糖和脯氨酸含量变化检测分析表明，枳落叶后抗性增强主要与脯氨酸增加有关。以早实枳和克里曼丁杂交F1代分离群体构建的高密度分子标记遗传图谱，进一步基于图谱和BSA分析两种手段，对落叶性状调控基因进行了QTL定位，将落叶性状调控基因定位到1号和8号两个染色体上1.307 Mb和0.67 Mb范围的两个区域，其中包含143个基因。进一步通过共分离检验、落叶前后表达量变化、基因组差异比较等手段对候选基因进行了筛选，对部分基因通过转基因及干旱、低温下处理进行了功能分析，推测磷蛋白ECP44可能参与了落叶执行过程，而FRI可能参与了落叶对低温与干旱的响应过程，但是该假设涉及的分子机理仍需要进一步研究加以证实。