Evaluation of Genetic Diversity of Plum Pox Virus in a Single Plum Tree

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For the first time, the analysis of the genetic diversity of Plum pox on a single plum tree
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/262823696 Evaluation of genetic diversity of Plum poxvirus in a single plum tree. Conference Paper  · August 2011 CITATIONS 6 READS 24 3 authors , including:Luká š  Predaj ň aSlovak Academy of Sciences 90   PUBLICATIONS   127   CITATIONS   SEE PROFILE Zdeno Š ubrSlovak Academy of Sciences 71   PUBLICATIONS   519   CITATIONS   SEE PROFILE All content following this page was uploaded by Zdeno Š ubr on 31 October 2014. The user has requested enhancement of the downloaded file.  L ukáš PREDAJŇA –    Alžbeta NAGYOVÁ –    Zdeno ŠUBR –   Miroslav GLASA Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 84505 Bratislava, Slovakia    Plum pox virus (PPV, genus  Potyvirus , family  Potyviridae ) is an aphid-borne RNA virus infecting perennial stone fruits of  Prunus  spp. crops worldwide [1]. Despite substantial progress in the ability to analyze and describe PPV variability, less attention is paid to understand the PPV diversity and evolution within single hosts. Dynamic genetic structure of virus populations has a significant role in the epidemiology of the virus, going up to the selection of variants with increased pathogenicity [2]. The fast mutation and large population size of RNA viruses produce populations of viral genomes known as quasispecies [3]. The quasispecies nature of PPV may imply a high adaptive potential, allowing for the rapid selection of biologically distinct variants with the highest fitness in new environments. A PPV-free two years old  Prunus domestica cv. Oullin ´ s Gage tree (grafted on St. Julien rootstock) grown in a pot under controlled conditions was triple inoculated in May 2003 by chip-budding with PPV-M (isolate VAR-2), PPV-D (isolate BOR-1) and PPV-Rec (isolate BOR-3) isolates. In spring 2004 the tree was replanted in an open field and let to develop untreated. In September 2010, seven years after chip- bud inoculation, the tree was sampled for analysis of PPV diversity. A 746-bp fragment spanning the C-ter NIb/N-ter CP region was amplified using the TaKaRa LA Taq polymerase (TaKaRa, Bio Inc.) and  primer pair NCuniFor 5 ′ -GAGGCAATTTGTGCTTCAATGG-3 ´  (sense) and NCuniRev 5 ´ -CGCTTAACTCCTTCATACCAAG-3 ´ (antisense). The RT-PCR products were directly sequenced and simultaneously, an aliquot of the same PCR products were cloned into the pGEM-T Easy cloning vector (Promega) and 7 randomly chosen cDNA clones were sequenced for each PCR product (in total, 105 individual clones were sequenced). Sequence analyses were performed using the Molecular Evolutionary Genetics Analysis (MEGA v.4.1) and DNA Sequence Polymorphism (DnaSP v.5) software. Conclusions: ã Sequence analysis revealed that after 7 years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and PPV-D) have been displaced. ã Existence of PPV genetic variability within single tree was demonstrated. ã Within PPV population, a total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely dominant. However, no clear-cut pattern of the viral population by the tree architecture could be highlighted. ã The intra-isolate variability observed for the PPV-M isolate was consistent with the quasi-species theory of RNA virus populations. References: 1.Garcia, J.A., and Cambra, M. 2007. Plant Viruses 1:69-79 2.Garcia-Arenal, F et al. 2001. Annu. Rev. Phytopathol. 39:157-186. 3.Domingo E. 2002. J. Virol. 76: 463-465 EVALUATION OF THE GENETIC DIVERSITY OF PLUM POX VIRUS IN A SINGLE PLUM TREE. This work was supported by the grant HUSK/0901/1.2.1/0126 and APVV-0042-10. 16 samples anaylsed from single tree.  From six seasonal shoots in different parts of the tree, 6 basal leaves (LB) and 6 apical leaves (LA) were collected. In addition, the skin of 4 developed fruits (F) from different parts of tree was also analysed. The alignment of the sequences of 15 PCR products showed that 9 of them were identical. The other 6 sequences each differed by a single point mutation. Only one substitution resulted in an amino acid change. The average pairwise nucleotide divergence between 15 master sequences was 0.098% (one sample being negative). Sample   Number of sequences/Number of haplotypes   Haplotype diversity (h)   Nucleotide diversity (Pi)   Average number of nucleotide differences (k)   F1   7/6   0.952   0.00312   2.190   F2   7/2   0.286   0.00081   0.571   F3   7/3   0.667   0.00149   1.048   F4   7/3   0.524   0.00122   0.857   LA1   7/3   0.524   0.00163   1.143   LA2   negative   LA3   7/5   0.857   0.00326   2.286   LA4   7/7   1   0.00393   2.762   LA5   7/4   0.714   0.00190   1.333   LA6   7/5   0.857   0.00244   1.714   LB1   7/4   0.714   0.00244   1.714   LB2   7/5   0.857   0.00285   2.000   LB3   7/3   0.524   0.00081   0.571   LB4   7/5   0.857   0.00204   1.429   LB5   7/5   0.857   0.00204   1.429   LB6   7/4   0.714   0.00163   1.143   whole dataset   105/51   0.879   0.00304   2.132   organ   Number of sequences/Number of haplotypes   Haplotype diversity (h)   Nucleotide diversity (Pi)   Average number of nucleotide differences (k)   Fruits (F)   28/12   0.836   0.00259   1.815   Apical leaves (LA)   35/21   0.864   0.00301   2.114   Basal leaves (LB)   42/22   0.892   0.00307   2.152   whole dataset direct PCR sequencing   15/4 0.600 0.00098 0.685  Analysis of the nucleotide polymorphism in the different samples depending of plant organ  Analysis of the nucleotide polymorphism in the different samples collected from single plum tree, F= fruit, LA= apical leaf, LB= basal leaf Since sequencing potentially leads to an underestimation of the extent and structure of PPV intra-host genetic diversity, the 15 PCR products were cloned and 105 individual cDNA clones were sequenced (7 clones per sample). The average pairwise genetic distance within this dataset was 0.304%, over three fold higher than the estimate obtained from directly sequenced PCR products. In total, the 105 sequences yielded 51 variants, two of which were highly predominant, representing respectively 29.5% and 19% of the sequences. The other 49 sequence variants were observed at significantly lower frequencies (0.9-3.8%). Successful inoculation of all 3 PPV isolates was documented by strain-specific RT-PCR one year post inoculation. From 2nd year on, only the PPV-M and PPV-Rec isolates could be detected, PPV-D no longer being detected. 5th year post inoculation only presence of PPV-M could be detected. Viewpublication statsViewpublication stats
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