黑龙江省和海南省稻瘟病菌中AVR-Pita家族的分布及变异分析

【目的】通过检测黑龙江省和海南省不同稻瘟病菌(Magnaporthe oryzae)菌株群体中AVR-Pita家族的分布情况和变异特征，了解其变异类型的致病表型，为区域内抗病种质的筛选与选育提供参考。【方法】通过参考NCBI中公布的AVR-Pita家族序列分别对启动子区和CDS区设计特异性引物共6对，对2020年采自黑龙江省和海南省不同地区的397个稻瘟病菌单孢菌株提取DNA，进行PCR扩增。通过电泳检测结果，分别选取囊括不同地区的代表菌株对扩增片段进行测序。测序结果与NCBI中相应的碱基与氨基酸序列进行比较分析，并利用水稻抗性单基因系，对不同变异类型的稻瘟病菌菌株进行无毒功能验证。【结果】在PCR电泳检测结果中，黑龙江省稻瘟病菌菌株携带所有待测基因，分布广泛且检出频率较高；而海南省稻瘟病菌菌株中仅携带AVR-Pita1和AVR-Pita2，并以低频率集中存在。无毒基因组成分析结果显示，黑龙江稻区的稻瘟病菌菌株复杂多样，携带的基因型种类较海南菌株丰富。PCR产物测序检测结果显示，AVR-Pita家族以点突变、插入和缺失为主要变异类型划分为19种,且不同稻瘟病菌群体来源的菌株，其变异类型...

Distribution and Variation Analysis of AVR-Pita Family in Magnaporthe oryzae from Heilongjiang Province and Hainan Province

【Objective】By detecting the distribution and variation characteristics of the AVR-Pita family in different Magnaporthe oryzae strains from Heilongjiang Province and Hainan Province, the pathogenic phenotypes of the variation types were understood, which provided a reference for the screening and breeding of disease-resistant germplasm in the region.【Method】Six pairs of specific primers were designed for the promoter region and CDS region by referring to the AVR-Pita family sequence published in NCBI. DNA was extracted from 397 single spore strains of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020, and PCR amplification was performed. By electrophoresis test results, representative strains from different regions were selected to sequence the amplified fragments. The sequencing results were compared with the corresponding base and amino acid sequences in NCBI, and the avirulence function of different variation types of M. oryzae strains was verified by using rice resistant single gene lines.【Result】In the PCR electrophoresis results, the M. oryzae strains in Heilongjiang Province carried all the genes to be tested, which were widely distributed and had a high detection frequency. However, only AVR-Pita1 and AVR-Pita2 were carried by M. oryzae strains in Hainan Province, and concentrated in low frequency. The results of avirulence gene composition analysis showed that the strains of M. oryzae in Heilongjiang were complex and diverse, and the genotypes were more abundant than those in Hainan. The sequencing results of PCR products showed that the AVR-Pita family was divided into 19 types with point mutation, insertion and deletion as the main variation types, and the variation types of strains from different M. oryzae populations were specific. Ten variation types were detected in AVR-Pita1, among which AVR-Pita1- (1-5) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita1- (6-10) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification of these ten variation types, it was found that the avirulence functions were lost. Eight variation types were detected in AVR-Pita2, among which AVR-Pita2- (1-4) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita2- (5-8) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification, it was found that the avirulence functions were lost. Only one variation type (AVR-Pita3-1) was detected in AVR-Pita3 in Heilongjiang Province.【Conclusion】The AVR-Pita family in Heilongjiang and Hainan populations were mutated alleles. After pathogenicity identification, all mutation types could not be identified by the corresponding resistance genes Pi-ta and Pi-ta². Therefore, the resistance genes Pi-ta and Pi-ta² can be used to polymerize other resistance genes to ensure the disease resistance of varieties in the process of disease resistance breeding and utilization of rice blast in Heilongjiang Province and Hainan Province. At the same time, the distribution and variation types of AVR-Pita family in M. oryzae strains from different geographical sources are specific.