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Agricultural Research Institute of the Hungarian Academy of Sciences
Detecting inter- and intraspecific recombination events in plant RNA viruses
with the TOPALI and RDP3 software packages
Gyöngyvér Gell, Endre Sebestyén, Ervin Balázs
Department of Applied Genomics, ARI-HAS, Martonvásár, Brunszvik str. 2, H-2462, Hungary
INTRODUCTION
Recombination among co-infecting plant RNA viruses is a
natural phenomenon that appears to have played a significant
role in the speciation and evolution of many strains. It also has
particular significance for the risk assessment of plants that
have been genetically modified for disease resistance by
incorporating viral sequences into plant genomes.
In the world of RNA viruses the source of recombination during
replication has a widely accepted model. By a process termed
‘template switching’, the viral replicase (an RNA dependent RNA
polymerase) might switch from its viral RNA template to
another viral RNA or to the transgenic mRNA and give rise to a
recombinant RNA molecule. Recombination has played a
significant role in the evolution of RNA viruses by producing
genetic variation, reducing mutational load, and introducing
new viruses (Worobey and Holmes, 1999). In the genus
Potyvirus, recombination has been reported for a number of
different species such as Plum pox virus (PPV) (Cervera et al.,
1993), Potato virus Y (PVY) (Revers et al., 1993), Bean common
mosaic virus (BCMV) (Revers et al., 1993), Yam mosaic virus
(YMV) (Bousalem et al., 2000), Lettuce mosaic virus (LMV)
(Krause-Sakate et al., 2003), and only one from the SCMV
subgroup, the Sugarcane mosaic virus (SCMV) (Zhong et al.,
2005).
Several diferrent software packages exist for the detection of
recombination between DNA and RNA sequences ‘in silico’. As
none of the statistical methods used by these softwares are
completely reliable and optimal for detecting recombination
under all conditions, we applied the PDM (Probabilistic
Divergence Measure) method from the TOPALi software
package and several other methods (RDP, GENECONV, BootScan,
MaxChi, Chimaera, SiScan, Phylpro, LARD and 3Seq) from the
RDP3 package.
RESULTS
Fig. 2.: The virion of MDMV.
Potyviruses form a group of long flexuous particles
composed of a single-stranded RNA (ssRNA) about
10 Kb long encapsidated by about 2000 copies of
capsid protein (CP).
Fig. 3 : MDMV genome organization
The single stranded RNA of (+) polarity is 9515 bp long
and carries a Vpg (viral protein genome-linked) covalently
bound to its 5’ end, and a Poly(A) tail at its 3’ end.
Fig. 4: Recombination analysis on the full length MDMV genomes
Left side: nucleotides between 1-2950. Right side: nucleotides between 2951-9563. The two phylogenetic trees are
significantly different, as detected by the TOPALi software, and this indicates a possible recombination breakpoint
in the region encoding the P3 protein.
Fig. 5.: One of the intraspecific recombination events in the full length genome of the Maize dwarf mosaic virus
detected with RDP3 (Recombination Detection Program). This breakpoint is located between the 1070th and
2707th nucleotides, in the region encoding the HC-Pro and P3 viral proteins.
CONCLUSION
Fig. 1: Chlorotic mosaic symptoms on the MDMV isolate Sz0603-M from Hungary.
The typical pattern of symptoms on maize is a mosaic extending the whole leaf blade margin. Lower leaves are often
free of symptoms. Plants are sometimes stunted to half their normal height with a reduction in ear size and seed set.
The TOPALi software found only one intraspecific
recombination event in the full length MDMV genomes (P3
cistron), while the RDP3 package detected 4 breakpoints
(1070-2707 nt-HC-Pro-P3, 3442-3786 nt-6K1-CI, 7215-7629
nt-NIb, 8668-64 nt-CP-5’UTR).
The analysis of the coat protein coding region of the SCMVsubgroup led to the detection of a large number of
interspecific recombination breakpoints (MDMV-MDMV;
MDMV-JGMV; MDMV-SrMV) with the RDP3 package but
none were detected with the TOPALi software.
In summary, our results demonstrate that recombination is a
major driving force in the virus evolution, and emergence of
new virus variants in the SCMV-subgroup, paired with
mutations, could generate altered biological properties.