Recombination plays a key role in the evolution of geminiviruses and may be contributing to the emergence of new strains/species. The high frequency of mixed infections of begomoviruses in different host allows the emergence of new viruses arising from recombination among species. With the development of computational recombination detection tools and an increasing number of available genome sequences, many studies have reported evidence of recombination in a wide range of Geminiviridae genera. The in silico analysis suggested that interspecific recombination has resulted significant diversity among geminiviruses and emergence of new geminivirus diseases.
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Geminiviridae is the largest family of plant DNA viruses that infect a broad range of plants causing devastating diseases for important crops (i.e., fruits, vegetables, ornamental plants and fiber crops (Morales and Anderson, 2001; Mansoor et al., 2003) and medicinal weeds (Prajapat et al., 2011a, b). Geminiviruses are characterized by circular single stranded DNA (ssDNA) genomes that encapsidated in twinned quasi isometric particles. The Geminiviridae family classified into four genera based on the genome organization and host range: Mastrevirus, Curtovirus, Topocuvirus and Begomovirus (Fauquet and Stanley, 2003; Van Regenmortel et al., 1999).
Begomovirus is the largest genus of the plant virus family Geminiviridae which members infect only dicotyledonous plants and transmitted by whitefly (Bemisia tabaci). Begomovirus those have bipartite genomes, comprising a DNA-A components required for replication and encapsidation and a DNA-B components required for virus movement and each of about 2.6-2.8 kb (Hanley-Bowdoin et al., 1999) or monopartite with all genes resident on one (DNA A-like) ssDNA of about 2.8 kb (Stanley et al., 2005).
The International Committee on Taxonomy of Viruses (ICTV) recommend taxonomic criterion for species of begomoviruses based on the applicability of these criteria to the large number of characterized begomoviruses. The comparative analyses of full-length DNA-A sequences were considered, based on recombination events that readily occur among begomoviruses (Pita et al., 2001; Fauquet et al., 2003). The nucleotide sequence identity of the A component up to 89% was established to distinguish different species from strains (Fauquet et al., 2003). Genomic organizations of begomoviruses are presented in Fig. 1.
|Fig. 1:||Genomic organization of begomoviruses: ORF positions and directions of translation are indicated by arrows in DNA-A and DNA-B (Wyant, 2011)|
Recombination defines as the exchange of DNA between similar DNA components and pseudorecombination is the exchange of DNA components (Polston and Anderson, 1997; Belete et al., 2011). The diversity of begomovirus species is associated with mixed infections, in which recombination and pseudorecombination events may explain the frequent emergence of new begomoviruses. Both events have been demonstrated in the laboratory (Garrido-Ramirez et al., 2000) and under natural conditions (Pita et al., 2001). Some isolates of Tomato Yellow Leaf Curl Virus (TYLCV), Potato Yellow Mosaic Virus (PYMV) and Mimosa Yellow Vein Virus (MYVV) are good examples of recombination and pseudorecombination (Pita et al., 2001; Monci et al., 2002; Prajapat et al., 2011b).
Variability of begomovirus species: Since recombination events showed in Begomovirus genus, partial sequences were not sufficient to distinguish new species (Fauquet et al., 2003). Studies to obtain complete sequences of the genomes of begomoviruses from appropriate samples to determine if they are indeed new species of begomoviruses. In addition recombination and pseudorecombination events can generate severe hybrids, as in the case of African Cassava Mosaic Virus (ACMV) (Pita et al., 2001). In less than 15 years ACMV isolates and species are no longer geographically distinct, the viruses are spread throughout the continent (Legg and Thresh, 2000; Pita et al., 2001).
Based on the phylogenetic analysis of viral sequences, it was proposed that, analogously to curtoviruses, Tomato pseudo-curly top virus (TPCV) resulted from recombination between mastreviruses and begomoviruses (Briddon et al., 1996; Palmer and Rybicki, 1998). A key factor in the genesis and spread of the pandemic was the recombination of two distinct cassava mosaic begomoviruses to produce a novel and more virulent hybrid (Pita et al., 2001).
In southern Spain, a natural recombinant between Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl (TYLCV) was detected and an infectious clone of a recombinant isolate (ES421/99) was obtained and characterized. Field studies revealed that the recombinant strain is becoming the predominant strain in the region in which it was detected (Monci et al., 2002).
In silico recombination analysis: Recombination between divergent genomes is a major mechanism by which diversity amongst viruses is generated (Robertson et al., 1995). Recombination Detection Program (RDP) utilized to detect the possibility of recombination in geminivirus isolates by using their sequence information which based on pair wise scanning approach. It runs under Windows 95/98/NT/XP/VISTA/7 and couples a high degree of analysis automation with an interactive and detailed graphical user interface.
The conclusions of recombination studies based on the evaluation of different methods of recombination detection (Posada and Crandall, 2001; Posada, 2002). The recombination breakpoint could be identified by using Recombination detection program [RDP] (Fig. 2), Geneconv (Fig. 3), Maximum-Chi (Fig. 4), Bootscan, Chimaera and 3SEQ methods (Table 1). All these methods were implemented in RDP v.3.44 (Martin et al., 2005). Default RDP v.3.44 settings were used throughout (Bonferroni correction and P-value), other than that sequences were considered as circular, consensus daughters were found and breakpoints were polished.
Natural virus recombinants: Newly emerging geminiviruses are causing severe disease epidemics in cotton, grain, legumes, tomato and other staple food and cash crops in tropical and subtropical regions of the world (Boulton, 2003; Khan, 2000).
|Table 1:||Different recombination detection methods available in RDP3|
|(RDP3: Instruction manual at http://darwin.uvigo.es/rdp/rdp.html)|
|Fig. 2:||An RDP pairwise identity plot for the piece of sequence from major parent (EU487045 AYVCNC) that breakpoint begin from 774th [position 1758 in alignment] position in alignment and ending breakpoint at position 94th in alignment of Mimosa Yellow vein virus (HQ876467). Approximate p-value for this region was 6.742x10-04.Uppermost bares indicating positions of informative sites, pink region indicates breakpoint positions suggested by the GENECONV method. The pairwise identity plot have major parent: minor parent plot (EU487045 AYVCNV: EU487048 ToLCCoV; yellow), recombinant: major parent plot (HQ876467 MYVMV: EU487045 AYVCNV; dark blue) and recombinant: minor parent plot (HQ876467 MYVMV: EU487048 ToLCCoV; purple)|
|Fig. 3:||The GENECONV plot of high scoring fragment (AJ888455) in recombinant Verbesina encelioides leaf curls alphasatellite (HQ631431). The approximate p-value was 6.581x10-16. In this case the left and right bounds of the pink region indicate breakpoint positions suggested by the GENECONV method|
|Fig. 4:||An example MaxChi plot of recombinant Ocimum leaf curl virus (JF968443) for beginning breakpoint position was 557th and ending breakpoint position was 54th in alignment. In this case the left and right bounds of the pink region indicate breakpoint positions suggested by the GENECONV method. Approximate p-values of two peaks were 1.062x10-02 and 8.435x10-04. Graph shows the schematic linearized map of putative recombinant fragments within the CP gene of the Ocimum sanctum begomovirus and related begomovirus isolates. Each horizontal line represents the genotype of one virus isolate and the color-coded boxes represent the tentative origins of the putative recombinant fragments|
These viruses cause a variety of symptoms in host plant species and are spreading at an alarming pace due to a high rate of recombination (Briddon et al., 2003; Mansoor et al., 2003). The rapidly evolve of begomoviruses, due to recombination and pseudorecombination (Hou and Gilbertson, 1996; Padidam et al., 1999). The emergence of new begomvirus strains / species in nature by recombination between previously existing species has been demonstrated (Zhou et al., 1997; Saunders et al., 2001).
It was observed that the different species of geminiviruses seem to recombine easily if infectious pseudorecombinant clones used (Unseld et al., 2000). In addition, examples of natural recombination have also been reported recently in cotton (Zhou et al., 1998; Sanz et al., 1999) and cassava (Zhou et al., 1997; Pita et al., 2001). In some cases, the new geminiviruses species those arising as a consequence of recombination, exhibited a new pathogenic phenotype which is often more virulent than the parents (Fauquet et al., 2005; Girish and Usha, 2005; Rojas et al., 2005; Garcia-Andres et al., 2006; Kon et al., 2006; Rothenstein et al., 2006).
Begomoviruses are considered to be an emergent group of plant viruses, due to the high incidence and severity of diseases caused by them over the last three decades, in tropical and subtropical regions of the world (Polston and Anderson, 1997; Legg and Thresh, 2000; Morales and Anderson, 2001; Briddon et al., 2003). Ageratum conyzoides plants exhibiting yellow vein symptoms, collected from China, contained begomoviral DNA-A like molecules. Sequence alignment shows that Ageratum Yellow Vein China Virus (AYVCNV) has arisen by recombination among viruses related to Ageratum yellow vein virus, Papaya leaf curl China virus and an unidentified begomovirus (Xiong et al., 2007).
Weeds can retain the virus that can be transmitted by the insect vector back to crop plants (Assuncao et al., 2006) causing yield loss of the crops. Additionally, because they act like virus reservoirs, recombination and generation of new viral genomes is facilitated (Frischmuth et al., 1997; Jovel et al., 2007; Morales and Anderson, 2001). Once present in the new host, these indigenous viruses would have rapidly evolved via recombination and pseudo recombination, giving rise to the species currently detected in the field (Castillo-Urquiza et al., 2008). The new geminiviruses originating from molecular recombination or pseudorecombination, as has been exemplified by Sida micrantha mosaic-associated viruses (SimMV). One of such viruses has developed recently and naturally by recombination between a DNA-A and a DNA-B components of different ancestors (Jovel et al., 2007).
The presence of multiple and recombinant betasatellites in Digera arvensis indicates that weeds could be important sources of multiple begomovirus components that affect crop plants. The presence of a recombinant betasatellite suggested that weeds are likely vessels for recombination and evolution of components of begomovirus complexes (Mubin et al., 2009). Rhynchosia minima (family Fabaceae) weed species exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, Mexico. Recombination analysis of the Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) genome indicated that the DNA-A component has arisen through intermolecular recombination (Hernandez-Zepeda et al., 2010). Therefore, the high frequency of mixed infections of begomoviruses facilitates the emergence of new viruses arising from recombination among strains/species (Harrison and Robinson, 1999; Power, 2000).
Viruses cause a variety of symptoms in host plant species and are spreading easily due to a high rate recombination and pseudorecombination events that contribute in the evolution of new virus strains/species. Inter-specific recombination has resulted in remarkable diversity among geminiviruses and that is the major cause of the emergence of new geminivirus diseases in tropical and subtropical regions. In some cases, the recombinants exhibited a new pathogenic phenotype which is often more virulent than the parents. With the development of reliable computational recombination detection tools and an increasing number of available genome sequences, many research reports have demonstrated evidence of recombination in a wide range of Geminiviridae genera.
This study could be used to understand the role of recombination and pseudorecombination in evolution of new Geminiviridae species and genetic diversity information could be considered for the planning of disease management strategies.
The authors are thankful to Department of Biotechnology (DBT), India and Department of Science and Technology, India for financial support for the present studies.
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