稻瘟病菌无毒基因AVR-Pi9在云南稻区的变异及微进化特点

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is the most devastating rice disease in the worldwide. 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 resistance gene Pi9 has extensively employed by breeder in China, while the variation of the corresponding AVR-Pi9 of M. oryzae is still unknown. In order to further conform the effectiveness and durability of Pi9 against to rice blast fungus. The objectives of the present study will be to determine DNA and protein variations of AVR gene(s) in selected field isolates from Yunnan rice production areas and to examine if these variations have any functional relationship with virulence reaction to corresponding R gene. Five hundred isolates of M. oryzae and 30 non rice fungus collected from Yunnan were selected as represent isolates. DNA will be isolated from mycelia of the 500 represent isolates of M. oryzae and 30 none rice blast fungus, and AVR-Pi9 genes primes were used to amplify the AVR-Pi9 gene allele and for sequencing, respectively. The DNA sequence variation of AVR-Pi9 avirulence gene will be analyzed. Meanwhile, the virulence of the 500 represent isolates of M. oryzae will be characterized on monogenic line (harboring resistance gene Pi9) and the susceptible variety Lijiangxintuanheigu(LTH), respectively. The virulence of each isolate on Pi9 resistance genes will be measured. 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. To declaimed the variation and evolution characteristics of AVR-Pi9 gene in rice blast fungus and non-rice blast fungus will be analyze, respectively. Through the above research, to declaimed the variation feature of the AVR-Pi9 genes and the molecular evolution mechanisms in field M. oryzae isolates population in Yunnan rice production areas; and to definite the distribution of AVR-Pi9 of M. oryzae population in rice growing regions of Yunnan. 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。前人研究推定云南是稻瘟病菌起源中心。Pi9基因被我国水稻育种者广泛利用，而AVR-Pi9的变异制机仍不清楚，为明确Pi9抗性持久性及有效性，本项目拟在前期工作基础上，对AVR-Pi9编码区及启动子区序列在云南不同生态区收集的稻瘟病菌株中选择出500个水稻菌和50个非水稻菌代表菌株中的变异进行分析；利用持Pi9抗性基因的水稻单基因系对菌株的毒性进行测定，分析AVR-Pi9基因编码区及启动子区的变异及其对Pi9基因的毒性的对应关系，明确AVR-Pi9编码区和启动子区在田间稻瘟病菌群体中的变异模式及分布特征；对敲除AVR-Pi9菌株的致病性和适合度变异进行分析，解析AVR-Pi9在水稻菌和非水稻菌中遗传变异及进化特征，揭示AVR-Pi9在云南稻区的变异及分子进化机制，为稻瘟病防控和水稻抗性基因的持续利用提供依据。

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。云南是稻瘟病菌多样性中心之一，也是亚洲栽培稻的分化中心，Pi9基因被我国水稻育种者广泛利用，而AVR-Pi9的变异制机仍不清楚，为明确Pi9抗性持久性及有效性，本研究通过分子技术和接种鉴定对稻瘟病菌无毒基因AVR-Pi9在云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：利用AVR-Pi9基因标记对云南稻区的不同寄主（包括水稻和非水稻寄主）上分离的梨孢菌菌株群体中的分布进行PCR检测，发现云南省不同稻区来自水稻的菌株均持有AVR-Pi9，而来自蟋蟀草、龙爪稷、马唐和香蕉的非水稻菌株中，只有来自蟋蟀草、龙爪稷的菌株持有AVR-Pi9，来自马唐和香蕉的菌株均不持有AVR-Pi9，说明AVR-Pi9在水稻和非水稻菌中的分布存在差异，AVR-Pi9在水稻稻瘟病菌株群体中十分保守，初步明确了AVR-Pi9在不同寄主（水稻和非水稻）上分离的梨孢菌群体中的分布特征；对AVR-P9的序列结构进行分析，分别发现了10个变异位点，揭示了梨孢菌菌群体中AVR-Pi9的变异形式；进一步对AVR-Pi9的单倍型进行分析，分别发现有8个单倍型，其中7个单倍型为水稻稻瘟病菌单倍型，1个单倍型只分布于蟋蟀草和龙爪稷上分离的菌株，构建了无毒基因AVR-Pi9的进化树，阐明了田间群体的进化趋势。为明确不同寄主菌株间在基因组水平的遗传差异，对8个分离自水稻、马唐、龙爪稷、蟋蟀草、香蕉的菌株进行了全基因组测序及比较基因组分析，发现来自香蕉和马唐的菌株基因组大小与参考菌株70-15相比相对较大，马唐和香蕉的分离菌株内发生了大片段插入和缺失突变，揭示了AVR-Pi9可能是通过遗传重组事件产生的能够被水稻等少数寄主识别的，在水稻分离菌中受到纯化选择的重要功能位点。.发表SCI研究论文1篇，已投在审稿论文2篇，国家发明专利受理1项。项目负责人获云南省中青年学术技术带头人和云南省专家基层科研工作站的进站专家。