基于SLAF-seq技术的四倍体蓝莓高密度遗传图谱构建及果实糖酸性状QTL定位

The blueberry is one of the most rapidly expanding fruits in China, and more prominent varieties are needed to meet the market requirement, which could adapt the soil and climate condition of China. Constructing the high density genetic linkage maps, dissecting the genetic basis of agronomic important traits and developing molecular markers for some elite loci are highly efficient on improving the blueberry breeding. In this project, the F1 progeniesbetween ‘Sapphire’ (southernhigh bush) and ‘Berkeley’ (northern high bush) were measured with soluble sugar and organic acid to clarify the distribution, segregation and inherit of these two traits. Meanwhile, we plan to perform high-throughput sequencing for the whole F1 population and their parents to develop SNP markers using SLAF technology, and construct the high-density genetic map of tetraploid blueberry based on SNP markers. The developed high density genetic linkage map will be applied in QTL mapping of soluble sugar and organic acid in blueberry, and the resultant QTL with large effects will be subjected to develop allele-specific PCR markers. These markers will be functionally evaluated with other bi-parental or natural population, which will improve the molecular breeding and understand the development of soluble sugar and organic acid in blueberry.

我国蓝莓产业快速发展，培育适应我国土壤及气候且品质优良的突破性品种来满足产业需求迫在眉睫。构建高密度的遗传连锁图谱并解析重要性状的遗传基础，建立重要位点的分子标记是加速蓝莓育种的有效途径。本项目以南高丛代表品种‘Sapphire’和北高丛代表品种‘Berkeley’构建的F1代为研究材料，通过测定可溶性糖和有机酸的含量和组成，明确这两个性状的群体分布、分离规律及遗传倾向；同时采用二代SLAF高通量测序技术开发多态性SNP标记，构建基于SNP标记的四倍体蓝莓高密度遗传连锁图谱。利用构建的遗传连锁图谱和糖酸等品质性状定位控制糖酸等性状的QTL，并对效应较大的QTL建立特异的分子标记，在其它杂交群体或自然群体中验证这些QTL的效应，为促进蓝莓的分子育种和解析糖酸性状的形成奠定基础。

【背景及科学意义】遗传连锁图谱是数量性状遗传基础解析和分子标记辅助育种（MSA）的基础，目前越橘属报道的遗传连锁图谱普遍存在标记少、密度低、集中在二倍体类型等问题，尚不能满足QTL定位及标记开发、基因挖掘等深入研究的要求，故构建四倍体高密度遗传连锁图谱，进而开展QTL定位、分子标记开发等研究，为推动蓝莓MSA与常规杂交育种相结合、提高育种效率奠定理论基础。【内容】本研究在解决蓝莓杂交种子萌发及作图群体真假杂种鉴定问题的基础上，基于大群体调查明确果实贮藏相关性状并分析其遗传趋势，通过Survey调研了解四倍体蓝莓基因组信息，利用SLAF高深度测序技术及基因组比对筛选的SNP标记构建同源四倍体蓝莓高密度遗传连锁图谱，对果实品质、贮藏相关性状进行QTL定位，筛选主要QTL位点。【结果】1）新鲜或自然阴干的蓝莓杂交种子，经400mg•L-1赤霉素浸种20h，采用上层水苔下层混合营养土的基质播种，种子萌发率达到85%以上。2）开发了一种基于SNP次等位基因频率，采用遗传关系分析和个体特有的稀有等位变异分析方法的非杂交后代鉴定方法，共鉴定出10个非杂交后代。3）蓝莓果实果蒂痕大小、果实硬度是与贮藏性相关的主要性状，其中果蒂痕大小遗传变异主要来自遗传效应，遗传潜能较大，果实硬度存在倾向于硬度变小的劣质遗传，遗传不稳定，易受环境影响。4）Survey调研预估‘Berkeley’单倍体核基因组大小541.95Mb，重复序列约占基因组序列的42.72%，杂合度为1.89%，属于高杂合的复杂基因组。5）基于高深度测序获得50455个高质量SNP标记，构建了同源四倍体蓝莓超高密度遗传连锁图谱，图谱包含12条连锁群，图谱总长2873.06cM，上图SNP标记数量达到37347个，标记间平均距离0.077cM，即12.99SNP/cM，图谱上仅存在4个标记距离≥5cM的gap。6）以LOD≥3.5作为QTL入选的临界值，共检测到41个与果实品质、贮藏性及果实大小等相关的QTLs位点，供试性状受多个QTLs控制，且多数QTLs表现为微效，年际间QTLs数量和在连锁群上的位置、表型解释率有所不同，但都存在稳定表达的QTLs。