辣椒抗青枯病主效QTL的精细定位及候选基因的功能分析

Bacterial wilt, incited by Ralstonia solanacearum, is a major soilborne vascular disease severely affecting pepper (Capsicum annuum) production worldwide. Dissecting the resistance molecular genetic mechanism against pepper bacterial wilt has important application value on resistance breeding. Lacking of high resistant material and accurate resistance evaluation is two of main bottleneck restricting on resistance genetic mapping of pepper bacterial wilt. Previously, we constructed a bioluminescent strain of R. solanacearum (BL-Rs7) marked with the lux operon, and developed a nondestructive method to measure invasiveness and spatio-temporal distribution of R. solanacearum. The utility of the bioluminescence assay was validated by comparing R. solanacearum infection dynamics in real-time in vivo between resistant line BVRC 1 and susceptible line BVRC 25. The distribution and multiplication of BL-Rs7 strain in resistant line BVRC 1 was conspicuously limited in plants inoculated in either roots or stem compared with susceptible line BVRC 25. A major QTL on chromosome 10 conferring resistance to R. solanacearum was identified by SLAF-BSA technology in resistant line BVRC 1. This proposal is intended to use bioinformatics analysis to develop SNP markers in major QTL region, and a large backcross population with precise resistance phenotypes will be used for fine mapping of the major QTL. Transcriptomes of BL-Rs7 strain infected pepper lines will be integrated to identify candidate resistant genes within QTL region, and the function of candidate resistance genes will be characterized by virus induced gene silence (VIGS) and transgenic technology. Furthermore, the functional molecular markers will be developed to lay foundation for large-scale screening resistant resources and establishing the system of molecular marker-assisted selection. The results will provide a valuable resource for preliminarily gaining insight into resistance mechanism between R. solanacearum and pepper plants.

由R. solanacearum引起的青枯病是辣椒生产中最严重的细菌性维管束病害。解析辣椒抗青枯病分子遗传机理对抗病育种具有重要的指导意义。缺乏高抗材料和精准的抗性评价是限制国内外抗青枯病遗传定位研究的主要瓶颈。为此，本课题组在国际上首次建立了一套生物发光标记青枯菌评价辣椒青枯病抗性的体系，实现了青枯病抗病表型的精准鉴定，并筛选获得一份可抑制青枯菌增殖和扩展的高抗自交系BVRC 1，通过对F2:3家系抗病表型的精准鉴定，基于SLAF-BSA技术将控制抗青枯病主效QTL定位在10号染色体12.69 Mb区间内。本项目将根据SLAF-seq测序结果和辣椒基因组信息，在主效QTL区间开发SNP标记，结合连续回交群体精准表型和基因型分析结果，对QTL所在区间进行精细遗传作图，克隆抗病候选基因，并利用病毒诱导的基因沉默和转基因技术验证其功能，为辣椒抗青枯病新品种选育提供技术支撑。

青枯病是辣椒生产中最严重的细菌性维管束病害，辣椒抗青枯病遗传定位研究进展缓慢，我国在该领域的研究甚少。在前期建立抗青枯病精准鉴定体系的基础上，本项目通过遗传分析发现BVRC1的抗性由不完全显性主效基因加微效基因控制，SLAF-BSA和QTL复合区间作图法将主效位点qRRs-10.1定位于10号染色体1.9-Mb区间，连锁标记ID10-194305124的抗性贡献率可达19%；在候选区间鉴定到5个抗病R基因可能与青枯病抗性有关。采用dual RNA-seq分析了BL-Rs7侵染BVRC 1时植株及菌转录组的动态变化，鉴定到48个随青枯菌增殖而协同持续上调表达的辣椒抗病相关基因，推测这些基因在抗青枯病反应中发挥重要功能；采用PacBio和Illumina测序相结合的方法组装和注释了青枯菌Rs-SY1高质量参考基因组，共注释出84个III型效应因子，其中5个III型效应因子在青枯菌侵染辣椒时持续上调表达，并构建相应的突变青枯菌株。通过对300多份辣椒材料的群体遗传分析，发现辣椒栽培种C. annuum主要按5种果形聚类，说明辣椒的驯化过程受到了果形选择的影响。前期对数百份辣椒材料的青枯病抗性评价，发现细线形的抗性相对较强，角形次之，而方形均高感；而且目前已知的抗病材料都为细线形或野生小果形。因此，推测辣椒青枯病抗病位点可能是从小果形向方形的驯化过程中被逐渐丢失。本研究为抗青枯病遗传改良和抗青枯病分子机制研究奠定了重要基础。