不同分布频率的3个稻瘟病菌无毒基因在西南稻区的变异及进化机制

Rice blast, caused by the ascomycete fungus M. oryzae, is the most devastating rice disease in the worldwide. This disease is effectively controlled by use of resistant rice cultivars. Blast R genes play a key role in monitoring invading fungus M. oryzae, recognizing the strains of the fungus that contain the corresponding avirulence (AVR) gene, and activating multifaceted defense mechanisms. Avirulence genes in M. oryzae are known to be highly variable and diverse. The high degree of variability and instability of AVR genes in M. oryzae often challenge the effectiveness of resistant cultivars with a single R gene a few years after their release. The objectives of the present study will be to determine DNA and protein variations of AVR gene(s) in selected field isolates from southwest rice production areas of China and to examine if these variations have any functional relationship with virulence reaction to corresponding R gene. Five hundred isolates of M. oryzae collected from southwest rice production areas of China were selected as represent isolates. In the previous research, we found that three avriulence genes, AVR-Pia, AVR-Pii and AVR-Pik with significant different distribution frequency in southwest rice production areas of China, the frequency of avirulence isolates to corresponding resistance gene are 17%, 33% and 55%, respecitively. DNA will be isolated from mycelia of the 500 represent isolates of M. oryzae, and three AVR genes primes were used to amplify the corresponding AVR gene allele and for sequencing, respectively. The DNA sequence variation of three avirulence gene will be analyzed, respectively. Meanwhile, the virulence of the 500 represent isolates of M. oryzae will be characterized on monogenic lines (harboring resistance gene including Pi-a, Pi-i, Pi-k, et al., respectively.) and the susceptible variety Lijiangxintuanheigu(LTH), respectively. The virulence of each isolate on each resistance genes will be measured, respectively. Base on above research, the relationship between the DNA sequence variation of avirulence gene and its virulence on the corresponding resistance gene will be analyzed, respectively. Through the above research, to declaimed the variation feature of the three avirulence gens(AVR-Pia, AVR-Pii and AVR-Pik) and the molecular evolution mechanisms in field M. oryzae isolates population of southwest rice production areas of China, respecitively; and to development the functional markers of three avirulence genes base on the functional variation of DNA sequence, respectively; To definite the distribution and compose of AVR genes in M. oryzae population in southwest rice growing regions of China. These results will provide useful foundation for sustainable use of rice resistance genes and the rice blast control.

稻瘟病是世界各稻区为害最严重的病害之一，无毒基因的变异是导致品种抗性丧失的主要原因。为了系统地了解在自然条件下稻瘟病菌无毒基因序列结构的变异及其与毒性的关系，本项目拟在前期工作基础上，在我国西南稻区收集的稻瘟病菌株中选择出500个代表菌株，对在该稻区分布频率存在显著差异的3个无毒基因AVR-Pia、AVR-Pii和AVR-Pik（分布频率分别为17%、33%、55%）的基因序列进行分析；同时，利用分别持有抗性基因Pi-a、Pi-i和Pi-k等抗性基因的水稻抗稻瘟病单基因系对菌株的毒性进行测定，分析无毒基因的变异及其对相应抗性基因的毒性之间的对应关系，揭示3个分布频率存在显著差异的无毒基因在田间稻瘟病菌群体中的变异特点及其在西南稻区的进化机制；开发相应无毒基因的功能性分子标记；明确3个无毒基因在西南稻区的组成及分布特征，为稻瘟病的防控和水稻抗性基因的持续利用提供理论依据。

稻瘟病是世界各稻区为害最严重的病害之一，无毒基因的变异是导致品种抗性丧失的主要原因。本研究通过分子技术和接种鉴定对稻瘟病菌无毒基因AVR-Pia、AVR-Pii和AVR-Pik在我国西南稻区稻瘟病菌群体中的变异及进化机制进行了研究，取得以下结果：分别利用AVR-Pia、AVR-Pii和AVR-Pik基因标记分别对AVR-Pia、AVR-Pii和AVR-Pik在西南稻区稻瘟病菌群体中的分布进行PCR检测，初步明确了这3个无毒基因在西南稻区的分布特征；对AVR-Pia、AVR-Pii和AVR-Pik的序列结构进行分析，分别发现了0、15和6个变异位点，揭示了田间稻瘟病菌群体中AVR-Pia、AVR-Pii和AVR-Pik的变异形式；进一步对AVR-Pia、AVR-Pii和AVR-Pik的单倍型进行分析，分别发现有1、3和10个单倍型，其中发现了AVR-Pii的2种新的单倍型和AVR-Pik的5种新单倍型，并发现1个AVR-Pii单倍型对Pi-i有毒，2个AVR-Pik单倍型对Pi-k/km/kp/kh有毒。以上结果表明，AVR-Pia的多态性为有无的变异，AVR-Pia基因通过完全缺失的方式，从无毒进化为有毒；AVR-Pii通过碱基的替换从无毒进化为有毒，而AVR-Pik位点（AVR-Pik/km/kp/kh）通过碱基的替换从无毒位点进化为有毒位点，揭示了AVR-Pii和AVR-Pik位点受到正向选择，AVR-Pik位点和AVR-Pii的突变是导致水稻小种抗性丧失的原因。利用进化软件分别构建了AVR-Pii和AVR-Pik基因进化树，发现AVR-Pii在西南稻区主要有2个进化分枝，而AVR-Pik位点（AVR-Pik/km/kp/kh）为逃避水稻抗性基因Pi-k位点（Pik/km/kp/kh）的识别，为阶梯式和网状进化模式。通过本研究，阐明了稻瘟病菌无毒基因AVR-Pia、AVR-Pii和AVR-Pik的在西南稻区的进化机制，将为稻瘟病的防控、抗性基因合理布局及水稻抗性基因的持续利用提供理论依据。.发表SCI研究论文1篇，已投在审稿论文2篇，获国家发明专利授权1项，国家发明专利受理1项。项目负责人被遴选为云南省中青年学术技术带头人后备人才培养对象和云南省专家基层科研工作站的进专家。