基于泛基因组学的柑橘黄龙病菌遗传变异与致病力分化研究

Citrus Huanglongbing (HLB) is a historical and widely distributed citrus disease in China. Previous study had revealed the genetic diversity of HLB-caused pathogen (“Candidatus Liberibacter asiaticus”, CLas) in China. However, the genetic variations of whole genome among CLas strains from different geographical locations and whether these genetic variations caused the pathogenicity differentiation remain unknown. In this project, the representative HLB-affected citrus samples are systematically collected from main HLB-epidemic areas in China. To evaluate and compare the pathogenicity of CLas strains, all CLas strains are maintained in different indicator citrus plants that show different resistance to HLB. Both the representative CLas strains from different geographical locations and CLas strains with different pathogenicity are sequenced to build the CLas genome database. The variation and similarity of whole genome sequence and genetic constitution among CLas strains from different regions are identified through Pan-genome analysis. The genetic variation and genetic diversity of CLas strains in China are analyzed based on Pan-genomic analysis result in genome level. In addition, the association analysis of pathogenicity differentiation result and genomic comparison result of different CLas strains is performed to identify the pathogenic genes resources that associated with pathogenicity differentiation. The function of candidate pathogenic genes is analyzed to reveal the genetic mechanism of CLas pathogenicity differentiation. Result from this project will not only help to understand the genetic variation and its relationship with pathogenicity differentiation among CLas strains, but also provides the theoretical basis for epidemic regularity and the pathogenic mechanism of citrus HLB.

柑橘黄龙病在我国发生历史悠久且分布广泛，前期研究表明我国黄龙病菌存在遗传多样性，但不同地区菌株间的全基因组遗传变异规律以及这些变异是否引起病菌产生致病力分化尚不清楚。本项目拟系统采集我国主要病区代表性黄龙病病原材料，选用不同抗性水平的柑橘品种为指示植物进行病原致病力测定与比较，以筛选表现不同致病力的菌株材料；同时对不同地区代表性菌株和表现不同致病力的菌株进行全基因组测序，构建我国黄龙病菌基因组数据库；利用泛基因组学分析不同地区菌株在基因组及基因组成上的差异和共性，进而从全基因组水平分析我国黄龙病菌的遗传变异和遗传多样性规律；同时关联分析不同菌株间的致病力差异与对应的基因组比较结果，筛选与致病力分化相关的病原基因资源并进行功能分析，以解析产生致病力分化的分子遗传机制。由此探明我国黄龙病菌的遗传变异规律及其与致病力分化的关系，为揭示黄龙病的流行规律和致病机制提供依据。

黄龙病是柑橘生产上最具毁灭性的病害，由候选韧皮部杆菌亚洲种所引起。前期研究表明我国黄龙病菌存在遗传多样性，但有关不同地区菌株间的遗传变异规律及是否产生致病力分化现象尚不清楚。本研究通过系统搜集我国黄龙病流行的10个省份共计132个菌株样本，进行全基因组测序与分析，并嫁接保存不同地理来源菌株材料30余份，初步构建了我国柑橘黄龙病菌病原资源库与全基因组数据库。主要结果如下：1. 通过对14个常见柑橘品种黄龙病样本的采集，系统分析了黄龙病菌在染病植株中的分布规律，即黄龙病菌在果实橘络组织中呈现富集，并以橘络作为基因组测序材料能显著提高黄龙病菌的基因组质量；2. 通过基于全基因组比较分析筛选高变异位点，并基于变异位点的扩大群体分析，对我国华南黄龙病流行区的病原遗传多样性进行系统探究，结果表明我国华南地区的黄龙病菌种群可分为三组（I组：广东、广西、湖南、福建及江西；II组：海南和浙江；III组：云南和贵州），阐明了我国黄龙病菌种群的遗传变异与地理位置存在紧密关联。3. 基于遗传变异与多样性结果，系统对比我国高海拔与低海拔黄龙病菌株的致病力差异，筛选了多个表现致病力差异的黄龙病菌株，对其中表现明显差异的代表菌株进行通过菟丝子-长春花体系进行致病力比较与分析，并进一步通过全基因组与互作转录组的比较分析，发现黄龙病菌在植物寄主中的增殖与致病表现与不同噬菌体类型存在很大关系，其中Type1类型噬菌体显著抑制病菌增殖从而影响黄龙病菌的致病表现，而携带Type2类型原噬菌体的黄龙病菌株则表现较强的致病力，包括涉及多个关键致病因子高表达的基因调控模式。本研究得到的结果，不仅初步解析了黄龙病菌遗传多样性与致病力差异间的关系与机制，而且为进一步探究CLas的致病机制提供了重要参考，具有重要的理论和实践意义。