进境木材腐朽菌小孔异担子菌KASP检测方法的建立  

Establishment of a KASP detection method for the root rot pathogen Heterobasidion parviporum on imported wood

作　　者：许剑涛 孙佳佳 赵鹏 杨静 潘能 刘晓宇 钱茱希 吴翠萍 李彬 蔡磊[3] XU Jiantao;SUN Jiajia;ZHAO Peng;YANG Jing;PAN Neng;LIU Xiaoyu;QIAN Zhuxi;WU Cuiping;LI Bin;CAI Lei(Animal,Plant and Food Inspection Center,Nanjing Customs,Nanjing 210001,Jiangsu,China;Taicang Customs Integrated Technical Service Center,Taicang 215400,Jiangsu,China;Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]南京海关动植物与食品检测中心,江苏南京210001 [2]太仓海关综合技术服务中心,江苏太仓215400 [3]中国科学院微生物研究所,北京100101

出　　处：《菌物学报》2024年第9期96-104,共9页Mycosystema

基　　金：国家重点研发计划(2023YFC2604904);海关总署科研项目(2020HK159);南京海关科研项目(2023KJ20)。

摘　　要：小孔异担子菌Heterobasidion parviporum是一种侵染性强、危害大的针叶树腐朽菌,在我国尚未分布,有随进境木材传入我国的风险。为建立小孔异担子菌的竞争性等位基因特异性PCR(kompetitive allele specific PCR,KASP)检测方法,本研究对收集的异担子菌属及其近似属的223条基因进行多序列比较分析,从小孔异担子菌GAPDH基因筛选出1个SNP位点作为检测标记,然后参照KASP引物设计要求设计引物Hpar-FAM-F1、Hpar-HEX-F2、Hpar-R,用于该分子标记的KASP检测。测试结果表明,利用开发的分子标记可以成功将小孔异担子菌与变孔异担子菌、西方异担子菌等22种近似种明确分型,说明该标记特异性好;灵敏度测试发现,将小孔异担子菌DNA浓度从100 ng/μL按10倍梯度稀释后,10^(-4)和10^(-5)稀释梯度的DNA反应结果与双蒸水接近,说明该标记的检测最低浓度可达0.1 ng/μL。阳性样品模拟分别使用担子果粉末与木材混合提取DNA和直接在木材样品中添加小孔异担子菌DNA进行KASP检测,结果表明,模拟阳性样品与参照近似种在坐标轴上表现出典型的基因分型,该KASP标记可以准确鉴定针叶木材上的小孔异担子菌。因此,本研究建立的KASP方法可用于检测进境针叶材上的小孔异担子菌。Heterobasidion parviporum is a strong devastating pathogen and great harmful to coniferous forests.It is not distributed in our country until now,but there is transmission risk of being introduced through imported wood.In order to establish a KASP detection method for H.parviporum,multiple sequence comparison was performed on 223 genes from Heterobasidion and its similar genus,and then a SNP site was screened on H.parviporum GAPDH gene,which can be used as marker in this study.The primers Hpar-FAM-F1,Hpar-HEX-F2 and Hpar-R were designed based on design rules for H.parviporum KASP molecular detection.Test results show that it could successfully identify H.parviporum from 22 similar species including H.irregulare,H.occidentale and others by genotyping,indicating that the marker has high specificity.DNA concentration of H.parviporum was ten-fold diluted from initial concentration 100 ng/μL in sensitivity testing.The reaction result of gradient 10^(-4) and 10^(-5) were close to that of double diluted water,and therefore,the minimum concentration of the marker detected was up to 0.1ng/μL.Model positive sample tests were performed in two forms,one was basidiocarp powder mixed with wood and another was adding DNA of H.parviporum in wood.Results show that model positive samples and the reference species displayed typical genotyping on axis.The KASP marker could accurately identify H.parviporum on coniferous wood,and the KASP method can be used to detect H.parviporum on imported wood.

关 键 词：小孔异担子菌 KASP标记 检测 

分 类 号：S41[农业科学—植物保护]