【作者】 高芳銮；

【导师】 谢联辉； 詹家绥；

【作者基本信息】 福建农林大学 ， 植物检疫， 2014， 博士

【摘要】 马铃薯Y病毒(Potato virus Y,PVY)为马铃薯病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)代表成员,是马铃薯和烟草生产中最为广泛并造成严重经济损失的病毒之一。迄今为止,有关我国PVY群体的遗传结构特征及其分子进化机制的研究鲜有报道。明确PVY群体遗传结构及演化机制,可为延缓马铃薯抗性品种使用寿命及可持续性植病防治奠定理论基础。1.本文对随机采自我国四大马铃薯耕作区16个省份的PVY疑似样品,采用间接ELISA方法逐一进行检测,并对其中的阳性样品材料通过电镜观察验证。PVY阳性样品经RNA提取后通过5对简并引物分别对P1、P3、CI、VPg和CP基因进行RT-PCR扩增、克隆、测序,获得的核苷酸序列分别进行遗传多样性的参数评估、群体分化检验、中性检验以及错配分布分析。遗传多样性分析结果显示,北方一作区(NSA)、中原二作区(CTA)、南方二作区(STA)和西南混作区(SMA)的单倍型多样性(Hd)均大于0.5、核苷酸多样性(Pi)均高于0.005,表明我国PVY群体具有高度遗传变异的特征。群体分化指数KST、Z和Snn评估结果显示,SMA与其他3个耕作区群体之间遗传差异显著,表明PVY群体在不同耕作区间存在较强的遗传分化。同时,中性检验结果表明,多数群体的Tajima’sD、Fu&Li’sD、Fu&Li’sF中性检验为负值,但仅有部分基因检验显著;而错配分布分析仅SMA的CP基因群体为单峰分布,这两方面的结果表明SMA的CP基因群体曾经历突然扩张的事件。2.为揭示突变、重组、自然选择和基因流等遗传力对我国PVY群体遗传多样性形成的影响,本文依次对这几个遗传力进行检测评估。变异分析显示,四个耕作区PVY群体的P1、P3、CI、VPg和CP基因的核苷酸序列一致性分别为72.10～100%。、79.20～100%、95.80～100%、82.40～100%和86.50～100%。其中,P1和P3基因的变异位点比例分别为52.06%和49.23%,而其他3个基因的变异位点比例为30.99～43.52%,表明P1和P3基因高度变异。重组分析显示,四个耕作区不同分离物的P1、VPg和CP基因均有重组现象。在这 5 个基因中,FEL(Fixed effects likelihood)、IEFL(Internal branches fixed-effects likelihood)、MEME(Mixed effects model of evolution)3 种方法均检测到大量的净化选择位点,说明PVY这5个基因许多突变都是有害的,在进化过程会被剔除。P3、CI和CP基因还检测到1～5个正向选择压力位点,表明这些变异有利于该病毒的生存竞争。同时,通过寄主和地区的适应性检验,总体上我国PVY的适应性进化同时受寄主和地区所驱动,说明我国的PVY群体同时具有寄主和地区特异性。基因流分析显示,除了NSA与SMA、CTA与SMA群体之间外,其他耕作区群体间存在着频繁的基因交流。综上结果表明,突变、重组、自然选择等遗传力是PVY进化的重要推动力。3.为揭示马铃薯Y病毒属(Potyvirus)的系统发育关系,本文根据该属已报道的pipo基因序列保守区设计一对简并引物,从感染PVY的马铃薯病叶中克隆获得pipo基因的cDNA全长序列,并基于氨基酸序列使用贝叶斯法重建了Potyviru 的系统发育树。系统发育分析结果显示,源于PVY的不同株系优先相聚成簇,而向曰葵褪绿斑驳病毒(Sunflower chlorottic mottlevirus,SuCMoV)与辣椒重型花叶病毒(Peppersevere mosaic virus,PepSMV)的亲缘关系较PVY相比更近,可以将原来以CP基因为分子标记误归入PVY组成员的SuCMoCV与PVY明显区分开来,表明pipo基因可以作为研究Potyvirus系统发育关系的新分子标记。同时,为实现PVY的快速检测和鉴定,本文以P1、HC-Pro、VPg和CP4个基因为研究对象,采用不同基因组合分别进行系统发育分析,并通过系统发育与性状关联分析进一步验证各种组合中代表分离物与株系的关联系数及相关参数,最终确定最佳组合以实现PVY主要株系的快速区分。结果表明,联合P1、HC-Pro、VPg和CP 4个基因系统发育分析能显著地将PVY已知株系的所有分离物区分开,而联合P1、VPg和CP3个基因,可以将PVY的常见株系区分开。4.为揭示多基因联合法检测到的一株疑似新重组分离物GF_YL20的全基因组结构特征,本文对该分离物进行了全基因组序列测定,并对其序列特征、重组位点、系统发育关系等进行了系统分析。GF_YL20分离物经克隆、测序和拼接获得的全基因组序列,不含有3’端的多聚腺苷尾端,序列长度为9,720nt,其中189-9,371 nt为编码区,编码一个3,061个氨基酸长度的多聚蛋白(Polyprotein)。序列分析显示,除了 VPg基因外,该分离物与PVYN:O株系的PB209分离物核苷酸序列一致性高达99%。但在VPg基因上,该分离物与PVYN株系的Mont分离物、PVYNTN株系的PB312分离物和SYR-I型的HN2分离物核苷酸序列一致性均超过97%。GF_YL20基因组内检测到3个显著的重组位点,这些位点不同于已知的PVY分离物的重组位点。系统发育分析显示,GF_YL20独立成簇而未聚于已知的P VY株系中。综合分析表明GF_YL20是一个新的PVY重组分离物。通过本文的研究,明确了我国马铃薯Y病毒群体的群体遗传结构特征,揭示了我国PVY的进化具有寄主特异性和地区特异性。同时建立了快速鉴定PVY株系的联合多基因体系,可为病毒病的防控奠定理论基础。更多还原

【Abstract】 Potato virus Y(PVY)is a type member of the genus Potyvirus of the family Potyviridae.It is one of the most destructive plant viruses,causing considerable economic losses to both potato and tobacco crops.To date,knowledge about the population genetic structure and evolutionary biology of the pathogen is limited,but is vitally important in developping sustainable management schemes and effectively using resistant genes.1.Potato leaves with PVY-like symptom were randomly collected from 16 provinces covering the four major potato cropping regions in China,including Northern single-cropping region(NSA),Central double-cropping region(CTA),Southern winter cropping region(STA)and Southwestern mixed cropping region(SMA)and evaluated with Enzyme-linked immunosorbent assay(ELISA)and a transmission electron microscope.Total RNA was extracted from leaves infected PVY and five genes(P1,P3,CI,VPg and CP)of PVY were amplified by reverse transcription-PCR(RT-PCR)using degenerate primers.Positive clones containing corresponding targeted genes were selected for nucleotide sequencing.Sequence data from five genes were used for estimation of genetic variation,population differentiation,neutrality test and mismatch distribution.Haplotype diversity and nucleotide diversity in the PVY populations from four cropping regions were all higher than 0.5 and 0.005,respectively.indicating that a high genetic diversity in these regions.KST,Z and Snn estimates of pairwise population differentiation between PVY populations form SMA and other regions were all significant,indicating a great spatial structure of the pathogen.Tajima’s D,Fu&Li’s D and Fu&Li’s F were all negative for the most populations but a few of them were significant.Only the SMA population displayed a unimodal mismatch in CP gene,suggesting that the population may have undergone a demographic expansion recently.2.Mutation,recombination,natural selection and gene flow events were estimated to determine the evolutionary forces responsible for the population genetic dynamics of PVY.Nucleotide sequence identities in P1,P3,CI,VPg and CP genes ranged from 72.10 to 100%,79.20 to 100%,95.80 to 100%,82.40 to 100%and 86.50 to 100%,respectively.PI and P3 had 52.06%and 49.23%polymorphic sites,respectively,suggesting the two genes are highly variable.Intragenic recombination was detected in PI,VPg and CP genes but not P3 and CI genes in the four cropping populations.Purifying selection were detected in the majority of polymorphic sites by fixed-effects likelihood method(FEL),internal branches fixed-effects likelihood method(IFEL)and mixed effects model of evolution method(MEME),suggesting that many of the mutations in the five genes are harmful and consequently eliminated by natural selection.Some polymorphic sites in P3,CI and CP genes were under positive selection,suggesting that these mutations were beneficial for PVY Significant population subdivision was detected in the viral populations originated from different hosts and geographic regions by Bayesian Tip-Association Significance(BaTS)test,indicating both host and local environment play an important role in the adaptation of the pathogen.In addition,the analyses of gene flow showed high levels of genetic flow among cropping regions except between NS A and SMA,CTA and SMA and indicated gene flow was also one of the important factors that shape the structure of PVY populations in China.Overall,mutation,recombination,natural selection and gene flow were all important in the evolution of PVY.3.To reveal the phylogenetic relationship in Potyvirus genera,the pipo gene of PVY was cloned using a pair of degenerate primers designed from its conserved regions and its sequences were used to reconstruct phylogenetic tree by a Bayesian inference method.This analysis reveals that PVY isolates are clustered in the same branch with high posterior probability,while Sunflower chlorotic mottle virus(SuCMoV)and Pepper severe mosaic virus(PepSMV)are closely related.However,SuCMoV was classified as a member of PVY subgroup when CP gene was used as a molecular marker.Results from this study suggests that the pipo gene could be a useful marker for phylogenetic analysis of Potyvirus genera.Additionally,to determine phylogentic relationship of strains within PVY species,isolate-strain association was evaluated using different combinations of concatenated sequences from P1,HC-pro,VPg and CP genes.The results indicated all strains can be distinguished using concatenated sequences from the four genes while the major PVY strains could be distinguished successfully using concatenated sequences from P1,VPg and CP genes.4.The full genome of GF_YL20,which probably is a new recombinant identified from multigene phylogenetic analysis,was sequenced and analyzed for recombination and phylogenetic relationships with other known strain.The complete sequence of the isolate has 9,720 nucleotides in length,excluding the poly(A)tail at the 3’end.It encodes a polyprotein of 3,061 amino acids and has a genomic structure different from previously reported PVY strains.The genome shares 99%nucleotide identity with PB209(PVYN:O)except for VPg,but more than 97%nucleotide identity with the VPg of Mont(PVYN),PB312(PVYNTN)and HN2(SYR-I).Three putative breakpoints were detected in the GF_YL20 genome with high confidence.Phylogenetic analysis indicates that GF_YL20 shares a common ancestral origin with the PVYN:O and PVYN-Wi strains but is divergent from other PVY strains.Taken together,these sequence analyses indicate that GF_YL20 is a novel NxO recombinant.Overall,this study extends our understanding in the population genetic structure and evolutionary processes in PVY.In addition,the establishment multigene phylogenetic analysis system facilitate the detection and identification of PVY strains.Taken together,results from this project provide usefull information and tools in designing sustainable PVY control strategies.更多还原