番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立  

Establishment of a KASP-SNP detection method for filamentous fungi Fusarium oxysporum f.sp.lycopersici and Fusarium oxysporum f.sp.radicis-lycopersici

作　　者：叶青静[1] 王荣青[1] 阮美颖[1] 程远[1] 万红建[1] 李志邈[1] 周国治[1] 姚祝平[1] Ye Qingjing;Wang Rongqing;Ruan Meiying;Cheng Yuan;Wan Hongjian;Li Zhimiao;Zhou Guozhi;Yao Zhuping(Institute of Vegetables,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,Zhejiang Province,China)

机构地区：[1]浙江省农业科学院蔬菜研究所,杭州310021

出　　处：《植物保护学报》2022年第3期879-889,共11页Journal of Plant Protection

基　　金：浙江省自然科学基金(LY19C150010);财政部和农业农村部国家现代农业产业技术体系(CARS-23-G44);国家重点研发计划(2017YFE0114500)。

摘　　要：为快速、准确地对番茄枯萎病菌Fusarium oxysporum f.sp.lycopersici(FOL)和番茄颈腐根腐病菌F.oxysporum f.sp.radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F.oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性(single nucleotide polymorphism,SNP)位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL_KASP、FOLrace1_KASP、FOLrace2_KASP和FOLrace3_KASP共4对分型清晰的引物。KASP-SNP技术对FORL、FOL-R1和FOL-R2的检测阳性率达100.00%,对FOL-R3的检测阳性率为92.68%;常规PCR方法对FOL-R1和FOL-R3的检测阳性率也达100.00%,而对FORL的检测阳性率只有59.09%。表明所建立的KASP-SNP技术可用于FOL-R1、FOL-R2、FOL-R3和FORL的快速、准确检测。To detect Fusarium oxysporum f.sp.lycopersici race 1(FOL-R1),race 2(FOL-R2)and race 3(FOL-R3)and F.oxysporum f.sp.radicis-lycopersici(FORL)of tomato quickly and accurately,a detection method based on the kompetitive allele specific PCR-single nucleotide polymorphism(KASPSNP)was established.The KASP-SNP specific primers were designed based on SNP locus of the exopolygalacturonase gene(pgx4).The reliability of the KASP-SNP markers was tested via comparing KASP-SNP method and routine PCR and DNA-sequence analyses of the internal transcribed spacer(ITS)region and pgx4 gene.The results showed that totally 35 SNP sites were found in FORL,FOLR1,FOL-R2 and FOL-R3,and 18 pairs of KASP-SNP primers were designed,among which four pairs of primers had been screened.The positive rates of FORL,FOL-R1 and FOL-R2 were 100.00%using KASP-SNP,and that of FOL-R3 was 92.68%.The positive rates of FOL-R1 and FOL-R3 were 100.00%using the routine PCR,but that of FORL was only 59.09%.This study demonstrated that KASP-SNP method can be used to detect FOL-R1,FOL-R2,FOL-R3 and FORL rapidly and accurately.

关 键 词：番茄 尖孢镰刀菌 KASP-SNP标记 分子鉴定 生理小种 

分 类 号：S436.412.1[农业科学—农业昆虫与害虫防治]