马铃薯Y病毒多基因系统发育分析及其在株系鉴定中的应用

摘要

为实现马铃薯Y病毒(Potato virus Y,PVY)常见株系的快速鉴定,本文以PVY的P1、HC-pro、VPg和CP4个基因为研究对象建立了快速准确的多基因联合体系.根据基因的不同组合建立5个不同数据集,分别进行系统发育分析,并通过贝叶斯标签关联显著性(Bayesian tip-association significance,BaTS)分析各数据集中代表分离物与株系的关联性,以确定实现PVY快速鉴定的最佳组合.不同数据集的系统发育及BaTS分析结果显示,除了联合P1、VPg和CP 3个基因数据集外,其他4个数据集均无法实现PVY常见株系的准确鉴定.采用不同建树方法对联合P1、VPg和CP 3个基因数据集比较分析显示,基于ML法和NJ法的系统发育树在拓扑结构上基本一致,均优于基于贝叶斯算法的最大分支置信(maximum clade credibility,MCC)树.同时,以HLJ26分离物为研究对象,对建立的多基因联合体系进行实际应用,结果显示该分离物与PVYNTN-NW株系的3个分离物SYR-Ⅱ-2-8、SYR-Ⅱ-Be1和SYR-Ⅱ-DrH以高置信值聚为一亚簇,表明该分离物可能属于PVYN~-NW株系(SYR-Ⅱ型).重组分析显示,HLJ26基因组存在4个潜在的重组信号,分别位于P1、HC-pro/P3、VPg和CP的5'-末端,与PVYNTN-NW株系(SYR-Ⅱ型)的重组位点相一致,表明其属于PVYNTN-NW株系(SYR-Ⅱ型).同时,应用多重RT-PCR成功扩增出约为1000 bp和400 bp的2个特异性片段,与PVYNTN-NW株系(SYR-Ⅱ型)的特异条带大小相一致.这些结果进一步支持了多基因联合体系的鉴定结果.联合P1、VPg和CP 3个基因数据集系统发育分析,可以实现PVY常见株系的准确鉴定.%The objective of this study is to develop a rapid and accurate multigene phylogenetic analysis to identify Potato virus Y (PVY) strains.The phylogenetic relationships of strains within the PVY species were evaluated with isolate-strain association using five datasets of concatenated sequences from the P1,HC-pro,VPg and CP genes to determine the best dataset for PVY strain identification.Results from phylogenetic analyses and Bayesian tip-association significance (BaTS) tests indicated that the major PVY strains could be distinguished using the P1,VPg and CP concatenated sequences datasets but not the remaining concatenated sequence datasets.Phylogenetic trees reconstructed from the concatenated sequences of P1,VPg and CP genes revealed that the ML and NJ trees had broadly similar topologies and that both were better than the maximum clade credibility tree (MCC).Additionally,the full genome of HLJ26,one isolate randomly selected for the multigene phylogenetic analysis,was clustered with high confidence among members of the PVYNTN-NW (SYR-Ⅱ) strain,which includes isolates of SYR-Ⅱ-2-8,SYR-Ⅱ -Bel and SYR-Ⅱ-DrH.This suggests that it was a PVYNTN-NW (SYR-Ⅱ) isolate.Recombination analysis of this isolate identified four putative recombination joints in the P 1,HC-pro/P3,VPg and the 5'-terminus of CP.This pattern is similar to that observed in the genomic structure of PVYNTN-NW (SYR-I),supporting the classification of this isolate as the PVYNTN-NW strain (SYR-Ⅱ[).Simultaneously,two expected fragments of approximately 1 000 and 400 bp in size were also amplified from the isolate by a multiplex RT-PCR,consistent with the expected band pattern of the PVYNTN-NW (SYR-Ⅱ) strain.This further supports the utility of the multigene phylogenetic method in identifying PVY strains.We propose that the major PVY strains could be distinguished accurately using multigene phylogenetic analysis based on the concatenated sequences from the P1,VPg and CP genes.