灌木柳耐盐相关基因鉴定及其连锁分子标记的筛选研究

As optimal material for study of forestry genomics, the shrub willow serves as an important source of bioenergy crops in the world. Despite several studies focused on selection of salinity tolerant clones in willow were carried out, the salinity tolerance genes were still untouched. Due to lack of functional molecular markers,the genomic study in willow has remained stagnant. In this project, salinity tolerant shrub willow was selected by salt water trial.Candidate genes were screened through RNA-seq by using of two samples under different stress treatments and cloned by RACE. Meanwhile, a set of EST-SSR markers were developed. A segregation population was constructed and the linkage analysis between molecular makers and salinity tolerance genes was detected by use of BSA way.After this work,the understanding of salinity tolerance mechanism was deeply enhanced in theory. The references for study of willow functional genomics can also be provided.And the global resources of willow genome will be significantly accumulated. On the other hand, the useful molecular markers were developed for resistance breeding. And the progress of willow breeding will be efficiently promoted in practice.

灌木柳是全世界重要的能源林营造树种之一，也是林木基因组研究的理想材料。虽然我国长期以来一直重视耐盐灌木柳无性系的选育，但耐盐基因方面的研究仍是一片空白。由于缺乏可靠的标记，柳树功能基因组学研究受到了极大限制。本项目拟以灌木柳为材料，通过水培试验筛选耐盐灌木柳植株，并利用RNA-seq 对不同胁迫处理下的样本进行高通量测序，筛选耐盐相关基因并进行全长克隆；利用测序序列开发一组EST-SSR标记；构建分离群体并利用BSA法进行标记与耐盐候选基因的初步连锁分析。本研究将在理论上加深林木耐盐分子机理的理解，填补林木耐盐基因组研究的空白，显著增强我国林木功能基因的资源积累；在应用上为柳树抗性育种提供可靠的功能标记，有效促进柳树分子育种的进程。

作为绿化沙漠、水淹地和盐碱地的重要树种，柳树已完成全基因组测序。为深入开展柳树功能基因组研究，探索柳树耐盐分子机制并加快分子标记辅助育种研究，本项目通过水培试验筛选出最耐盐灌木柳无性系“2345”。并以清水为对照，在高盐胁迫下0h、2h、6h、12h、24h及48h为时间节点，取上、中、下部叶片及根毛分别构建混合文库进行转录组测序。经生物信息学分析统计出表达差异基因信息并进行功能注释。挑选出肌醇磷酸合成酶基因（MIPS），设计引物并进行全长克隆。实时荧光定量PCR结果显示柳树SjMIPS 基因在171mM NaCl盐胁迫处理24-48h后上调，表明柳树MIPS基因是盐胁迫诱导型基因，在参与肌醇的生物合成、调节渗透压方面起着重要作用。同时基于GenBank中EST序列、垂柳与簸箕柳转录组拼接序列以及耐盐无性系“2345”转录组拼接序列，利用MISA软件进行位点查找，分别开发EST-SSR标记8个、4521个及7175个，并对其中部分位点进行了通用性及多态性验证。通过子代分离群体的构建，选取2个亲本及最耐盐与最敏盐的子代个体各50个进行简并基因组测序，开发10万个SLAF标签并找出在这两个极端混池中表现出多态性的标记，对关联出来的区域，结合物种的参考基因组序列，做功能注释，找到与耐盐基因相连锁的分子标记。本项目筛选柳树耐盐相关候选基因并克隆其全长，显著增强了我国林木功能基因的资源积累；开发的大量genic-SSR 标记，为后期柳树基因组学的研究提供有力的技术支持；开展分子标记与耐盐候选基因的连锁分析，有效的促进了柳树分子育种进程，也为其他性状和其他树种提供了有益的参考。