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. 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. CONCLUSION The TOPALi software found only one intraspecific recombination event in the full length MDMV genomes (P3 cistron), while the RDP3 package detected 4 breakpoints ( nt-HC-Pro-P3, nt-6K1-CI, nt-NIb, nt-CP-5’UTR). The analysis of the coat protein coding region of the SCMV- subgroup 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. 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 Right side: nucleotides between 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. 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. 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. RESULTS