草莓通过花瓣迅速脱落逃避灰葡萄孢侵染的机制研究

Strawberry is widely cultivated in China with its high nutritional and economic value. Botrytis fruit rot (BFR), caused by the necrotrophic fungus Botrytis cinerea, is the main disease of strawberry during its growth and storage period and no strawberry variety is highly resistant to BFR. Strawberry fruits were infected by B. cinerea via the senescent petals attached to the surface of the fruits. Senescent petals provide B. cinerea perfect nutrition for infection. In the three years of pre-study with 33 strawberry varieties, the applicant found that there was a significant negative correlation between the speed of strawberry petal abscission and disease incidence of BFR. The variety "Sweet Charlie" had a slow speed of petal abscission (SAB) and a high incidence of BFR, while the "Red Cheek" had a fast speed of petal abscission (FAB) and a low incidence of BFR. The ability of Red Cheek to avoid attack of BFR is disease escape, due to separation of fruits and spores by quick abscission of petals. To understand the mechanisms related to the speed of petal abscission, this project will analyze the differences between the groups of SAB and FAB from the aspects of organizational structures, metabolism and gene expression. Bulked Segregation Analysis will be used to establish the SAB and FAB pools. Histological differences will be observed with ultra-thin slices. The metabolic and genetic differences will be analyzed using GC-MS, LC-MS and RNA-seq. SNPs and SSR markers closely linked with fast petal abscission will be obtained. The results will be helpful for elaborating the physiological and molecular mechanisms of fast petal abscission in strawberry and provide a theoretical basis for breeding and development of new strategies for the control of BFR.

草莓是我国广泛种植的重要水果。由灰葡萄孢侵染引起的果实灰霉病是草莓生长期和储藏期的主要病害，目前尚没有发现高抗灰霉病的草莓品种。灰葡萄孢是一种非专化性死体营养型真菌，需以衰老花瓣为中介侵染草莓果实。项目申请人前期经过连续三年田间观察发现，不同草莓品种间花瓣脱落速度存在显著差异，果实灰霉病发病率与花瓣脱落速度显著负相关。通过使衰老花瓣迅速脱落，避免果实感染灰霉病，这是一种避病性。但是，导致不同草莓品种花瓣脱落速度存在显著差异的机制尚不明确。本申请项目拟采用集群分离分析法建立花瓣快脱落(FAB)与慢脱落(SAB)库，采用超薄切片、GC-MS、LC-MS和RNAseq等技术对FAB与SAB进行分析比较，明确草莓花瓣快脱落性状的组织结构和生理机制。基于全长转录组序列进行SSR和SNPs分析，建立与草莓花瓣快脱落性状紧密连锁的分子标记。项目的完成将为抗灰霉病育种及发展防治灰霉病的新策略提供理论依据。

本项目研究了草莓通过花瓣快速脱落逃避灰葡萄孢侵染的机制。. 首先，本研究建立了以花瓣迅速脱落为基础的草莓灰霉病抗性评价方法，对139份国内外草莓种质资源花瓣脱落速度进行了评价。结果显示：供试草莓种质资源的花瓣脱落性状存在显著差异，现蕾7天后花瓣脱落率介于3.33%-100%，花瓣脱落速度的性状不呈正态分布。脱落慢的占比较少，花瓣脱落速度极慢（<10%）的品种数占总测试品种的2.16%；脱落快的占比多，脱落率大于80%的品种占41.73%，花瓣脱落速度极快（100%）的品种比例为18.71%。..其次，明确了影响花瓣脱落快慢相关的组织结构因素。研究发现，草莓开花后存在萼片往回收缩的过程，花瓣脱落快的品种（‘红颜’）的花瓣在萼片收缩之前已形成离层，因此容易脱落；而花瓣脱落慢的品种（‘甜查理’）的花瓣在萼片收缩之后才形成离层，因此非常容易被收缩的萼片包裹住，不易与果实脱离接触。本研究从组织结构、激素变化、基因表达三个层面对草莓花瓣快速脱落的机制进行了解析。结果显示：‘红颜’在花开放后1d时花瓣连接部位的细胞排列疏松出现了离层，在2.5d时花瓣连接部位脱落；‘甜查理’在花开放后1d-2.5d时花瓣连接部位的细胞排列疏松，在3d时花瓣连接部位出现离层并脱落。激素含量及相应基因表达变化趋势均显示在开花初期‘红颜’的花瓣中ABA、乙烯含量均高于‘甜查理’。..最后，利用‘红颜’与‘甜查理’进行杂交，得到F2分离群体，对群体亲本及极端性状混池分别进行BSR测序。从中筛选出5个与花瓣迅速脱落相关联位点，分别位于染色体2-2（0-2.01Mb）、4-4（0.14-0.64）和6-3（39.16-40.22Mb; 40.36-40.40Mb; 40.66-40.68 Mb）上。本研究挑选出了14个候选基因，将其SNP转换成KASP分子标记，在资源群体中验证标记与表型的相关性，其中Fvb2-2_776356和Fvb2-2_619967的标记检测结果与花瓣脱落快慢呈极显著相关，相关系数分别为0.713和0.597。..项目研究结果为灰霉病的抗病育种奠定了理论基础。项目资助发表中文核以及以上论文4篇，其中SCI收录2篇。

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