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

【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 the multi locus genotypes(MLGs)of Psa for all primer combinations;and using the entire genome sequences of 20 and 10 Psa strains,a UPGMA clustering tree was constructed using the bruvo.boot()function based on the selected primer combinations to verify the typing effect of the selected primer combinations.【Results】34 pairs of primers had good amplification efficiency for Psa in China.By analyzing the results of simulated PCR data,it was found that TR14 and TR11II,TR19 and Psa-01 had the same sequence.TR8 and Psa-08,TR39II and Psa-10,GM-1834 and TR10I,GM-1553 and TR64II,TR19Psa-01 and TR19II amplified the same TR.The TR unit length of Psa-09 amplification product was not unique and the lateral variation of TR2II amplification product was large,which was not determining TRs by electrophoresis.By calculating the MLGs of each primer typing result,it was found that 34 pairs of primer MLGs were between 2-20.Among them,TR10Ⅰ,GM-1834,TR39Ⅱ,and Psa-10 had the highest MLGs of 20,while TR15I,TR17 and TR22 had the lowest MLGs of 2;By calculating the SI of each primer typing result,it was found that the SI values of 34 pairs of primers ranged from 0.0154 to 0.8968,with TR10Ⅰ,GM-1834 having the highest SI value of 0.8968 and TR15I having the lowest SI value of 0.0154.Through the program developed by R,it was found that the SI value of different primer combination typing results using all primer combinations was 0.9847.The combination of two pairs of primers with the highest SI was Psa03 and GM-1834TR10I,with an SI value of 0.9736,which did not reach the SI value of all primers.The combination of three pairs of primers with the highest SI was Psa03,GM-1834TR10I,and TR39IIPsa10,with an SI value of 0.9803,which still did not reach the SI value of all primers.The primer combinations with the same SI values as all primers were TR23/Psa-04,Psa-03,Psa-05,Psa-06,TR10IGM-1834,TR3II,TR1II,Psa-10TR39II and TR64IIGM-1553,among which TR23 and Psa04 can be replaced with each other,and you can choose one of them.The MLGs of the 9 primers typing results were equal to the MLGs of all primer typing results,UPGMA cluster tree analysis found that 9 pairs of primer combinations can accurately separate the 5 biovar of Psa,and some differences within the biovar 3 can be seen.【Conclusion】The above results indicated that using a combination of these 9 primers for typing analysis had the same effect as using all primers.After verification,the combination of 9 pairs of primers can accurately separate the 5 biovars of Psa.The combination of 9 pairs of primers including TR23/Psa-04,Psa-03,Psa-05,Psa-06,TR10IGM-1834,TR3II,TR1II,Psa-10TR39II and TR64IIGM-1553,can be used to study the population genetic structure of Psa,explore the transmission and prevalence patterns of kiwifruit canker disease,and provide scientific basis for the formulation of disease prevention and control strategies.