鸭茅抗锈病分子机制研究及抗性主效基因定位克隆

Orchardgrass (Dactylis glomerata L.), as a significant forage grass, plays an important role in stock raising and in management of the rock-desertification of southwest China. However, orchardgrass is sensitive to rust disease under high temperature and humidity, resulting in serious effect on its yield and quality. The molecular mechanism and major rust resistance genes of rust resistance are critical scientific issues, but they remain unclear. Based on the previous studies, the high-throughput sequencing for mRNA and miRNA will be used to discover the differential expressed genes in resistant (PI325293) and sensitive (02681) orchardgrass under the rust infecting, and expression quantitative trait Loci (eQTL) analysis will be conducted by using these different expressed genes to deeply reveal the rust resistant molecular mechanism for orchardgrass. And then the rust resistance is identified and evaluated in multi-plot for several years for the genetic linkage group and associated mapping population. The genotypes of GBS-SNP of whole genome and SSR were identified to conduct the rust resistant genome-wide association analysis (GWAS) and quantitative trait locus (QTL) to dig out molecular markers and sequences closely-linked resistant trait. Finally, one or two major rust resistant genes of orchardgrass will be cloned by synthesizing the data from QTL, GWAS and RNA-Seq analysis. The results will provide significant rust resistant materials, basic data, and technical supports, and can also accelerate the orchardgrass utilization in our country and rust resistant molecular breeding process.

鸭茅是世界重要牧草，在我国西南区草牧业及石漠化治理中发挥重要作用，但在高温高湿环境下极易感染锈病，严重影响其产量和品质。鸭茅抗锈病的分子机制及主效基因挖掘是鸭茅抗锈病分子育种关键科学问题，但目前尚未清楚。本项目拟对已筛选出的高抗锈病（PI325293）和高感锈病（02681）材料，通过mRNA、miRNA深度测序，挖掘鸭茅在锈病侵染下的差异表达基因，并对其进行表达数量性状基因座（eQTL）分析，深度解析鸭茅抗锈病的分子机制。再利用已构建的关联及连锁作图群体开展多年多点抗锈病鉴定评价和GBS-SNP、SSR基因分型，并进行抗锈病的全基因组关联（GWAS）及数量性状位点（QTL）分析，挖掘抗锈病分子标记及序列片段。最后综合QTL、GWAS和RNA-Seq数据克隆1-2个鸭茅抗锈病主效基因。研究结果可为鸭茅抗锈病分子育种提供重要抗性材料、基础数据和技术支撑，加快我国鸭茅资源开发利用进程。

鸭茅是世界著名的禾本科多年生冷季型牧草，蕴含有丰富的优异基因资源，因此在牧草生产中发现其具有高产、优质且适应性强等特点。在西南地区草地畜牧业、人工草地建植、生态环境治理中鸭茅也被广泛使用，然而在实际生产中发现鸭茅对锈病的易感性严重制约了其生产和利用。因此，挖掘鸭茅自身抗锈病基因资源，培育利用持久高抗品种是最根本有效的防治措施。本研究通过全基因组关联分析，联合转录组测序和miRNA测序进一步分析了鸭茅抵御锈病胁迫相关的分子机制。主要研究结果如下：.1）将所有材料种植于雅安、重庆（荣昌）两地，对感病后的锈病表型进行鉴定，筛选得到了10份优异研究材料；.2）对所有材料进行简化基因组测序（GBS)，通过分析锈菌胁迫相关SNPs标记，进行关联分析挖掘到了34个候选基因；.3）对材料中抗性极端材料进行转录组和miRNA测序，结合降解组测序分析发现40个与鸭茅锈菌胁迫相关miRNA以及38个对应的靶基因，并对其中miR156、miR172和miR394进行了互作验证，对3个miRNA及其对应靶基因进行锈菌接种后表达趋势分析，发现miR156与靶基因（DG0G01071.1）在锈菌接种后3天左右具有明显的差异表达，而miR394与靶基因（DG7G00548.1）在5天后才响应锈菌刺激，有趣的是，miR172在锈菌接种后从1天到13天表达量显著降低，而其靶基因（DG7G04366.1)在接种后从1天到5天表达量持续增加。.4）前期我们发现较多的WRKY基因在锈病胁迫下差异表达，对所有WRKY和RGA进行WGCNA分析，通过双荧光素酶报告基因系统体外验证发现有8对基因间存在互作关系，通过酵母单杂交技术进一步验证得到DG6C03650.1能与WRKY50相互作用。.以上研究结果将为进一步开展鸭茅分子标记辅助选择育种（MAS）、抗病性定向改良以及抗病品种培育，提高鸭茅育种水平和育种效率奠定了重要的理论基础。