巨桉抗枝瘿姬小蜂候选基因的筛选和关联分析

Eucalypts are sound species for establishing industry-oriented plantations in tropical and subtropical regions all over the world, but gall wasp (Leptocybe invasa), a pest reported in the recent decade, has prompted a great threat to their development. Eucalyptus grandis is the second tree species (after Populus trichocarpa) whose whole genome sequence has been released, and discovery of functional genes including those related with pest resistance will be the focus of studies in the post-genomic era. In this proposal, we intend to discover the candidate genes related with gall wasp resistance in E. grandis using RNA-seq and digital gene expression profiling techniques, develop single nucleotide polymorphism (SNP) markers with the candidate genes, and, in respect of the genetic structure of E. grandis populations, identify the SNP association with and the magnitude for the resistance phenotypes. In addition, the gene functionality will be verified through quantitative RT-PCR method. This will give insights into the molecular mechanisms for the interaction of tree and parasitic gall wasp, and increase significantly the tree functional genomics resources in China. Also, the functional markers developed herein will have potentials in practical molecular breeding in gall wasp resistance of Eucalyptus species and be valuable for accelerating molecular breeding in other economic traits.

桉树是重要的工业用材树种，但近年发现的枝瘿姬小蜂严重危害桉树人工林。巨桉是第二个完成全基因组测序的林木，包括抗虫在内的功能基因挖掘将是后基因组时代的重要内容。本项目拟通过RNA-seq和数字表达谱分析筛选巨桉抗枝瘿姬小蜂的候选基因，开发候选基因的SNP标记；结合巨桉群体的遗传结构分析、进行候选基因SNP与抗性的关联分析，发现抗虫的基因型、阐明抗虫基因型的表型效应，并通过RT-PCR验证抗虫基因型的功能表达。这将在理论上加深对林木-寄生蜂互作的分子机理的理解，显著增强我国林木功能基因的资源积累；也将在应用上为桉树抗枝瘿姬小蜂的分子育种提供可靠的功能标记，有效促进桉树分子育种的发展。

桉树是重要的工业用材树种，但近年发现的枝瘿姬小蜂严重危害桉树人工林。巨桉是第二个完成全基因组测序的林木，包括抗虫在内的功能基因挖掘将是后基因组时代的重要内容。本项目基于散布于全基因组的微卫星(SSR)标记，分析了巨桉天然群体的遗传结构，检测了与年均温、等温性和年降雨量等关联的受选择位点(Sci Rep, 2016, 6: 34941)；并通过与巨桉群体抗枝瘿姬小蜂的关联分析，检测到了7个显著关联的SSR位点(P<0.05)，单个位点解释的变异率为3.3~37.8%，其中4个SSR关联位点在细叶桉中得到验证(DNA Res, 已投稿)；通过抗虫和感虫基因型以及感虫前后共12个样品的RNA-seq测序，共获得48.16 M的高质量读序、总碱基47.19 Gb，数字表达谱分析筛选出与巨桉抗枝瘿姬小蜂相关的候选基因21条；并对巨桉群体进行了21个候选基因的扩增子测序(测序平台PacBio RSII)，检测了单核苷酸多态性(SNP)标记及其与抗枝瘿姬小蜂的关联，鉴定了显著关联的6个基因、27个SNPs (P<0.05)。此外，针对二倍体PCR产物直接测序的SNP检测，开发了检测软件DiSNPindel (BMC Bioinformatics, 2015, 16: 343; 发明专利号ZL 2013 1 0611263. 1)；结合关联验证的细叶桉群体，分析了与原产地气候因子相关的受选择SSR位点[林业科学, 2016, 52(9): 39-47]以及与早期生长相关的SSR位点[分子植物育种, 2016, 14(1): 195-203]。这是植物中首次开展的致瘿昆虫抗性相关的基因组位点的研究，理论上有助于加深对林木-寄生蜂互作的分子机理的理解，显著增强我国林木功能基因的资源积累；也将在应用上为桉树抗枝瘿姬小蜂的分子育种提供可靠的功能标记，有效促进桉树分子育种的发展。. 在本项目的资助下，已发表研究论文5篇，其中2篇发表在SCI源刊物；获得授权的发明专利1项；培养博士研究生1名、硕士研究生1名。