豇豆特有基因挖掘及其耐旱关联等位变异的鉴定

Originating from the tropic and sub-tropic regions of Africa, cowpea ranks one of the most drought tolerant cultivated crops worldwide. Previous studies have indicated some unique mechanisms underlying the inherent drought tolerance of cowpea and highlighted the involvement of species-specific genes in this process. Supported by the NSFC fund last round, we have identified dozens of drought candidate genes, part of which are found to be cowpea specific genes also, demonstrating the rationale to investigate the relationship between drought tolerance and cowpea specific genes at a genome scale. In this project, we plan to first of all predict cowpea specific genes by comparing unigene sequences of cowpea and the genomes of model legumes, followed by CGH validation. A custom exon-capture chip will then be developed to isolate all identified cowpea specific genes from the mini-core population and high throughput sequencing will subsequently be employed to identify natural sequence variations. Through the GWAS approach, the sequence variations associated with drought tolerance will be discovered. In parallel, a spatial transcriptional profiling across diverse tissues, a comparative analysis of the transcriptome in roots and leaves between normal and stressed plants as well as prediction of the interaction between cowpea specific genes and drought related miRNAs will be performed, to gain an insight into the role of cowpea specific genes in drought tolerance from a regulatory perspective. The final goal is to identify drought candidate genes and their favorable allelic variations that are unique to cowpea and associated with drought tolerance, which will be valauble to broaden our knowledge on the mechanisms of drought tolerance in cowpea. This work will also solidify the basis for genetic improvement of crops by taking advantage of cowpea specific genes/mechanisms.

豇豆起源于非洲，是耐旱性极强的栽培作物。前人研究表明豇豆的耐旱性具有其独特性并与物种特有基因有关。申请人在上一国家基金资助下鉴定了一批豇豆耐旱候选基因，亦发现若干基因为豇豆特有，为从特有基因入手系统探究其与超强耐旱性的关系提供了进一步依据。本项目以对豇豆unigene数据库中豇豆特有基因的挖掘和鉴定为切入点，通过对微核心种质群体进行外显子捕获来定向挖掘特有基因的DNA序列自然变异，并通过关联分析鉴定这些变异与耐旱性的关联。另一方面，通过不同组织及干旱胁迫下根和叶中豇豆特有基因的表达谱分析辅以特有基因-干旱调节的miRNA互作预测，从调控角度进一步分析豇豆特有基因与超强耐旱性的关系。通过整合两方面结果，挖掘出若干候选的与豇豆超强耐旱性有较直接关系的特有基因及其等位变异。本研究有助于了解豇豆超强耐旱性形成机制，可为探索利用豇豆超强耐旱基因改良作物耐旱性提供理论支撑，具有一定的前沿性和探索性。

越来越多的证据表明物种特有基因与生物特异性状有关。本研究中我们通过生物信息学结合CGH芯片杂交验证，鉴定了578条豇豆特有基因转录本。统计分析表明豇豆特有基因转录本具有长度短、GC含量低等特征。与普通基因转录本相比，特有基因表达量明显偏低，且呈现明显组织特异性表达。有677个基因在4种组织中均有表达，在花中特异表达的基因是根中的2倍。比对豇豆基因组发现特有基因呈成簇分布。有15个豇豆特有基因被预测与microRNA存在互作。应用序列捕获+高通量测序方法从8个豇豆品种中捕获测序了572个特有基因序列。经序列比对、SNP calling等在390个特有基因上鉴定了1560个多态SNP位点。利用LGC SNP检测平台，从上述SNP侧翼序列中设计了379个荧光PCR检测标记，并对223份豇豆种质进行了基因型检测。利用2012年和2014年年豇豆耐旱性表型数据进行关联分析，鉴定了26个显著位点。.项目执行期间发表标注项目资助的 SCI 论文 2 篇（其中1篇IF > 4.0），国内核心期刊论文1篇，待投稿SCI论文1篇，获国家发明专利授权1项。研究成果在亚洲动植物基因组大会（2016年，新加坡）上进行口头报告交流。2014年项目负责人赴美国加州大学开展合作研究。项目培养在读硕士研究生 1 人，项目负责人徐沛晋升为研究员，并于2014年入选中组部青年拔尖人才。