利用分离群体序列组装定位紫菜薹花青苷合成基因

Purple Tsai-tai (Brassica rapa L. ssp. chinensis var. purpurea) is a very popular vegetable, which is rich in anthocyanin. Identifying anthocyanin synthesis genes (ASG) is important for increasing genetically anthocyanin content in purple Tsai-tai. The whole genome assembly of B. rapa provides a reference for determining the ASG. However, our previous study shew significant difference between the reference and the genome of purple Tsai-tai. There is a great risk of missing the purple Tsai-tai specific ASG when using reference based methods. To solve this problem, we plan to identify ASG in purple Tsai-tai using de novo genome assembly based on segregation population sequencing. First, generate sequencing data of purple Tsai-tai segregation population and then perform "contig assembly - - high density linkage map construction - - chromosome concatenating" to complete the genome assembly. Second, perform the QTL analysis and screen the candidate genes with the linkage map and the genome sequences. The implement of this project will lay the foundation for the breeding of purple Tsai-tai with high anthocyanin content. Furthermore, the low cost de novo assembly strategy developed here is a universal method appliable in other species and will have broad influence on genomics.

紫菜薹富含具有保健作用的花青苷，是深受人们喜爱的蔬菜。确定紫菜薹花青苷合成基因（紫色基因）对提高其花青苷含量、改善其营养品质具有重要意义。白菜基因组测序完成为紫菜薹紫色基因的鉴定提供了参照序列。但是，前期研究显示紫菜薹与白菜参照基因组之间序列差异极大，依赖参照基因组的方法极可能漏掉紫菜薹特异的目标基因。为此，本项目拟建立利用分离群体测序数据的de novo组装方法解决上述问题。首先，在获得紫菜薹分离群体测序数据基础上，通过"Contig组装-高密度遗传图谱构建-染色体拼接"实现亲本基因组的序列组装，然后利用高密度遗传图谱和基因组序列进行紫色性状QTL定位和候选基因鉴定。本项目的完成，不仅为培育高花青苷含量的紫菜薹打下基础，而且建立了一种通用的低成本基因组组装方法，对基因组学研究具有广泛意义。

按照项目总体目标,完成了紫菜薹和菜心的基因组测序组装。对两个亲本进行测序得到约64X数据。组装获得紫菜苔基因组大小为306Mb,Scaffold N50为345Kb;菜心组装基因组大小为304Mb,Scaffold N50为526Kb。构建紫菜薹和菜心的 F2 分离群体,完成82份F2单株的DNA提取并完成了低覆盖度测序。进行遗传变异检测, 并构建高密度遗传图。在10条染色体上共发现969个bin标记,利用这些bin标记构建连锁图,得到了总遗传距离为926cM的高密度遗传图。利用本实验室自主开发的基因注释流程,对紫菜薹基因组进行基因注释, 最终预测出来 40732个基因。通过 QTL 分析在 A09 连锁群上发现控制紫菜薹花青苷积累的主效 QTL位点,定位于 1.9Mb 的区域内。在定位区域中找到了拟南芥中 AtEGL3 的共线性基因 BrEGL3.1 和 BrEGL3.2,重测序分析发现 BrEGL3.1 在亲本间存在较大变异,可能为控制紫菜薹花青苷积累的候选基因。该结果将为揭示白菜花青苷合成代谢机制的提供依据,而且为通过分子育种的方法培育富含花青苷的白菜新品种奠定基础。