Molecular Detection Of Narcissus Yellow Stripe Virus And Its Population Genetic Structure

Narcissus is a precious floral plant in China that has a long history of exportation.In recent years,narcissus virus diseases occur frequently and have major impacts on the quality and yields of narcissus.However,However,the researches of the detection and molecular evolution on narcissus viruses are limited,but vital in understanding the epidemic and evolution of narcissus viruses,and developing the sustainable management schemes.1.To investigate the occurrence of narcissus virus disease in China,RT-PCR was used to detect the infected narcissus leaves collected from various cultivation areas during 2016 and 2018.The results from RT-PCR revealed that 69.20%,58.75%,52.37%and 35.28%were positive for narcissus yellow stripe virus（NYSV）,narcissus degeneration virus,narcissus late season yellows virus and narcissus common latent virus,respectively.In addition,mixed infection of multiple viruses frequently occurred in the planting region of narcissus production in China.2.NYSV is one of the most destructive viruses that causes considerable losses to narcissus production.To develop a fast and accurate multi-gene-based RT-PCR detection assay for NYSV,two pairs of specific primers were designed from the conserved regions of NYSV cytoplasmic inclusion protein（CI）and coat protein（CP）,respectively.The multi-gene-based PCR detection assay was established after optimizing parameters including concentration of annealing temperature of the primers.The specificity and sensitivity of the assay were also tested.In addition,the utility of the assay was tested by detecting NYSV in plant samples infected by this virus.The optimized reaction system we established is:cDNA 2μL,CP258-F/CP258-R each 1.375μL（the primer pair of CP gene,10μmol/L）,CI510-F/CI510R each 0.625μL（the primer pair of CI gene10μmol/L）,2×PCR Master Mix 12.5μL,dd H₂O 6.5μL,annealing temperature 50℃,and for 35 cycles.The specific expected fragments of 258bp and 510bp gene fragments were amplified by this multi-gene-based RT-PCR detection assays.Specificity and sensitivity of the primer pairs were tested,and the total DNA was diluted to 10^-4.The multi gene-based RT-PCR detection assay has strong specificity,high sensitivity,excellent repeatability,which is useful in the detection and identification of NYSV in port quarantine.3.Genetic diversity parameters and population differentiation were estimated by analyzing nucleotide sequences of the CP and CI genes,sampled from Shanghai Chongming（CM）,Fujian Pingtan（PT）and Fujian Zhangzhou（ZZ）between 2010 and 2019.Mutation and natural selection were also assesed to determine evolutionary forces responsible for the population genetic dynamics.Meanwhile,Bayesian Tip-association Significance testing（Ba TS）were used to reveal the role of the geographic factors in the adaptive evolution of NYSV.K_ST,S_nn and F_STestimates of pairwise population differentiation were all significant（P<0.001）,indicating a great spatial structure of the pathogen.Negative selection was detected in the majority of polymorphic sites both in the CI and CI genes,although some polymorphic sites were under positive selection.In Ba TS analysis,the viral isolates from the same population tended to group together,showing a distinct geographic feature.Migraion analysis showed that strong gene flow was also contribute significantly to the population genetic dynamics of NSYV populations.Taken together,these results suggest that geography-driven adaptation was accounted for the population subdivision of NYSV and mutation,natural selection and gene flow all contribute to the population genetity and dynamics of the virus.4.Complete genomic sequences of five NYSV isolates（ZZ49,PT39,CM1,Ta1 and Ne1＿1）were sequenced and analyzed for their sequence properties,phylogenetic relationship with NYSV and NLSYV,as well as recombination.The complete sequences of these isolates have 9318nucleotides（nt）in length,except 9321nt for Ta1 islate.All isolates were grouped into a cluster together with other NYSV isoates,whereas Ta1 and Ne1＿1 were clustered with NLSVY isolates when reconstructred phylogenetic tree using the complete sequence.Sequence analyses showed that sequences identities of some coding region of these isolates were beyond the threshold of virus classification between NYSV and NLSYV.In addition,complicated recombination signals were detected in all 5 isolates.Overall,the results obtained indicated a complex evolutionary connenction between NYSV and NLSYV and the species demacraion between these two viruses should be optimized.In summary,we investigated the occurrence of narcissus virus disease in China and developed a multi-gene-based RT-PCR detection assay for NYSV in this study.Meanwhile,we characterized the genetic structure of three NYSV populations in China and identified molecular evolutionary forces responsible for the population differentiation in NYSV.In addition,we revealed that the NYSV diversification was driven by geography-specific adaptation.Taken together,the results obtained from this study will advance our understanding the evolution of NYSV and pave the way of the development of more effective management strategies.