猕猴桃溃疡病病原菌MLVA分型引物的筛选及验证  

Screening and validation of MLVA typing primers for the Pseudomonas syringae pv.actinidiae

作　　者：姚令 王海[2] 黄露[1] 安星宇 陈文 王莉爽 薛原 吴石平[1] YAO Ling;WANG Hai;HUANG Lu;AN Xingyu;CHEN Wen;WANG Lishuang;XUE Yuan;WU Shiping(Institute of Plant Protection,Guizhou Academy of Agricultural Sciences/Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Mountains Ministry of Agriculture and Rural Affairs,Guiyang 550006,Guizhou,China;Institute of Agricultural Science and Technology Information,Guizhou Academy of Agricultural Sciences,Guiyang 550006,Guizhou,China;Anshun Branch of Guizhou Tobacco Company,Anshun 561000,Guizhou,China)

机构地区：[1]贵州省农业科学院植物保护研究所·农业农村部喀斯特山区作物基因资源与种质创新重点实验室,贵阳550006 [2]贵州省农业科学院农业科技信息研究所,贵阳550006 [3]贵州省烟草公司安顺市公司,贵州安顺561000

出　　处：《果树学报》2024年第6期1188-1198,共11页Journal of Fruit Science

基　　金：贵州省科技计划项目(黔科合基础[2019]1308号)。

摘　　要：【目的】筛选出一组可精准快速地对丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv.actinidiae,Psa)进行分型的引物组合。【方法】针对前期文献已报道的34对引物,采用PCR技术验证该34对引物对中国Psa菌株的扩增效率及准确性;利用模拟PCR获取菌株串联重复(TR)数;以辛普森指数(Simpson’s index,SI)作为筛选引物组合的标准,基于R软件平台,筛选最优引物组合。【结果】34对引物对中国Psa扩增效果均良好,其中TR14与TR11II、TR19与Psa-01引物序列相同;TR8与Psa-08、TR39II与Psa-10、GM-1834与TR10I、GM-1553与TR64II、TR19Psa-01与TR19II扩增同一TR;Psa-09扩增产物串联重复单元长度不唯一,TR2II扩增产物侧翼变异较大,不能通过电泳确定串联重复数;最终确定SI值与全部引物组合相同的最低引物数量为9对,使用该9对引物的组合可将Psa已知的5种生物型准确分开。【结论】TR23/Psa-04、Psa-03、Psa-05、Psa-06、TR10IGM-1834、TR30I、TR1II、Psa-10TR39II、TR64IIGM-1553等9对引物可代表当前文献报道的34对引物,进行Psa分型研究,探索猕猴桃溃疡病的传播和流行规律,为病害防控策略的制定提供科学依据。【Objective】Kiwifruit canker caused by Pseudomonas syringae pv.actinidiae(Psa),is one of the most threatening diseases in the kiwifruit industry.Studying the population genetic structure of Psa can provide theoretical reference for scientific prevention and control of this disease.The multiple-locus variable-number tandem-repeats analysis(MLVA)has been reported to study the population genetic structure of Psa.At present,there are problems with inconsistent and excessive primers in the analysis of the genetic structure of Psa population using MLVA technology.Using too many primers will make MLVA typing technology lose its advantages like convenience and low cost.In order to screen primer combinations suitable for studying the population genetic structure of Psa,34 pairs of primers reported were analyzed.【Methods】To verify the amplification efficiency of primers on Chinese Psa,we used 34 pairs of primers to amplify 10 strains of Psa isolated and preserved in our laboratory.We downloaded the whole genome data of 127 Psa strains from Genbank for primer screening.We then used 34 pairs of primers on the genome sequences of these 127 Psa strains to perform Simulated PCR to obtain TR data.Calculate MLGs and SI using“popper”package to evaluate the typing ability of each primer with Simpson index(SI)as the standard for screening primer combinations,develop a program code using R to calculate the SI of typing results for different primer combinations,and use the df2genind()function of the“poppr”package to convert the simulated PCR result data into genind format.Primer combination SI calculation was performed by using the diversity_stats[mlg.table()]function,starting from the SI of the typing results of 2 pairs of primer combinations,and then calculating 3 pairs of primer combinations until the SI of the calculated primer combination was equal to the SI of all primers.This primer combination was the optimal primer combination.Use the genotype_curve()function of the“poppr”package to statistically analyze th

关 键 词：丁香假单胞菌猕猴桃致病变种 多位点串联重复序列分析 群体遗传结构 引物 筛选 

分 类 号：S663.4[农业科学—果树学] S436.634[农业科学—园艺学]