基于高通量SNP标记和关联分析整合分析油菜磷高效QTL及候选基因研究

Phosphorus (P) is an essential macronutrient for plants. Traits for P efficiency in plants are often controlled by multiple genes scattered throughout the genome. Brassica napus is an important oil crop in China, which has high yield and high P requirement but is sensitive to P deficiency in environment. However，Brassica napus has a developed root system for strong P-uptake ability from soil without proteiod root and mycorrhiza. In our previous research, quantitative trait loci (QTLs) of P efficiency in B. napus was conducted using two permanent mapping populations. In the present project, we use a set of newly developed single nucleotide polymorphism (SNP) markers distributed to the whole genome to constructed a high-density genetic map, and then use the map to perform a genome-wide QTL detection for P efficiency in B. napus. Genome-wide association mapping by high throughput SNP marker is aslo to be used to confirm QTLs/loci identified from both mapping populations. Based on the sequence information of molecular markers and the geome sequence of B. rapa, B. oleracea and B. napus, important candidate genes are to be predicted in the QTL intervals controlling P efficiency in B. napus. Candidate-gene association mapping is to be employed to validate main-effect genes for P efficiency in B. napus. Finally, 1-2 candidate genes will be isolated from different P-efficient cultivars in B. napus. Structure, temporal and special expression analysis, and Arabidopsis transformation of the candidate genes will be conducted to unravel the molecular mechnism of P efficiency in B. napus. Through this project research, we hope to meta-analysis B. napus P-efficient QTLs and their candidate genes, and to reveal the genetic mechnism of P efficiency in B. napus. This project will provide important theoretical basis and research strategies for genetic research of P efficiency in B. napus and other crops with complicated genome.

磷是植物生长发育所必需的大量营养元素。植物磷营养高效为复杂的数量性状，受多基因的调控。油菜是我国重要的油料作物，吸收利用土壤磷的能力较强，但是需磷较多，对缺磷敏感。本项目在前期油菜磷高效QTL定位的基础上，以不同磷效率种质及其发展的永久作图群体为材料，利用全基因组SNP标记图谱整合分析苗期磷高效QTL。同时以油菜自然变异群体为材料，开展基于高通量SNP标记与苗期表型的全基因组关联分析。二者相互验证，鉴定油菜磷高效的主要QTL/位点。在此基础上，通过生物信息学分析预测磷高效QTL区间的重要候选基因，然后利用候选基因关联分析方法鉴定候选基因的功能。最终获得1-2个重要的油菜磷高效候选基因，通过基因结构、序列分析、时空表达差异、转化拟南芥等研究初步探明基因的功能，揭示油菜磷高效的遗传机理，为油菜及其它复杂基因组作物磷营养性状的遗传研究提供理论基础和研究策略。

磷是植物生长发育所必需的大量营养元素。植物磷高效受多基因的调控，是复杂的数量性状。油菜是我国重要的油料作物，吸收利用土壤磷的能力较强，但是需磷较多，对缺磷敏感。本项目在前期定位的大量油菜磷高效数量性状位点（quantitative trait loci，QTL）的基础上，利用QTL比较分析、全基因组关联分析及转基因等技术，开展了油菜磷高效候选基因的克隆及功能初步研究。按照项目申请书的设置，我们开展了各项研究工作，完成了主要研究内容，取得了预期的研究成果。具体结果包括：1）利用高通量SNP标记构建了一张含2041个分子标记高密度的TN DH遗传连锁图，其中包含1698个SNP bin。利用该图谱进行了磷效率相关QTL的重新定位，共获得131个QTL。利用甘蓝型油菜物理图谱进行比对找到了135个QTL可以在TN DH群体和BE RIL群体中重复定位；2）利用含189个品种的油菜自然群体在两个磷水平下开展了四批苗期营养液培养试验，调查了群体在不同磷水平下的干重、磷效率系数等表型，结合群体3,824,552个SNP/Indel标记进行了全基因组关联分析，共获得171个与油菜苗期磷效率关联的SNP/Indel位点。将连锁分析定位的QTL与关联分析获得的SNP/Indel位点进行比对，在油菜全基因组共找到102个位点与苗期磷效率相关；3）在磷高效位点间找到24个候选基因，最终确定甘蓝型油菜4个BnaCRF8s转录因子基因作为目标基因，分离了基因的全长序列，并通过生物信息学分析研究了基因的进化、保守结构域、理化性质、蛋白质二级结构等；4）基因的时空表达模式分析表明4个BnaCRF8s基因均受缺磷诱导，但模式有所差别。在拟南芥中超表达BnaCRF8s基因会抑制植株根系的生长，进而影响对磷的吸收。转录组共表达分析表明4个BnaCRF8s与超过2,000个基因存在互作关系。本项目研究结果为揭示油菜磷高效的分子机制提供了新的理论基础。