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Natural sequence rearrangements in plant viruses are one of the keys in evolutionary processes determining the genomes architecture. Quantitative estimation of the recombination effect is decisive to our understanding about genetic diversity and its dynamics in the population. This present study was aimed to uncover the genetic basis for disease, and the evolution of virulence and pathogenicity by viruses. In this study, we mainly emphasized on Begomovirus's species from India and adjoining countries. Mainly detection of recombination and phylogenetic relations has been evaluated among the virus reported from the respective countries. The results have been derived based on information and data analyzed using RDP2, a bioinformatics tool used for detection of recombination in the sequences. Of the 956 sequences used in this study, 36 were shown highest degree of recombination. The Tomato yellow leaf curl viruses, namely Y184488.1; AM709505.1; EU573715.1 and DQ272543.1 were identified with the highest number recombination event. Mungbean yellow mosaic India virus (AY939925.1) was found to be in high risk with 1170 recombination event. The phylogenic analysis was done among the highly recombinant sequences which have shown evidence of viral evolution in Begomoviru's genus. We have attempted to evaluate risk of evolution of new strains trough recombination, in this region.
Keywords; Begomovirus, Recombination, Tomato yellow leaf curl, split tree analysis
During the course of evolution, Natural selection is typically viewed as the most commanding evolutionary force; however, it does not create new genetic variants. The mutation and the novel recombination events are the major factors to generate new genetic variants among population or texon. Detection of recombination among viruses was as a consequence of phylogenetic incongruence observed during identification using similar methodology use for bacteria and humans. Natural sequence rearrangements in viral genomes, as well as experimentally induced recombination events, both demonstrate the viruses have effective means of rearranging their genome. Natural rearrangements have been observed for the several plant viruses, like majority of sunhemp mosaic virus's (SHMV) genome is very similar to tobacco mosaic virus (TMV) while the organization of its 3' parts bear clear similarity to, otherwise unrelated, turnip yellow mosaic virus (TYMV). Recombination has been documented to occur between related and unrelated viruses as well as between DNA and RNA viruses (Gibbs et al., 1999; Worobey, 2000; Posada, et al., 2002; Meshi et al., 1981). The rate of recombination is generally influenced by selection limitation on the size and stability of the genome and the inevitability for increased genetic diversity (Chare & Hollmes 2006). In this scaffold, the functions of recombination are to purge the genome of accumulated lethal mutations as well as to increase diversity by dispersing valuable combinations of mutations. Recombination also provides genetic stability by tidy up the mutations potentially facilitating defective strains to recoup function (Lefeuvre et al., 2009). The study suggests that recombinants can achieve greater fitness, this potentially generates the genomic modifications, which affect virulence and pathogenicity and need to be critically evaluate the evolution (Deng et al., 1997; Fauquet & Stanley, 2003, Hudson & Kaplan 1985; Lazarowitz 1992; Moflat, 1999).
Begomoviruses have a wide host range and their genomes evolve fast, and they have caused significant yield losses to many crops, such as tomato, cassava and cotton in 39 countries (Saeed, 2010). The begomovirus are unique group of infectious agent that replicate and cause disease in a wide variety of the plant species, including many of agricultural importance. In addition to their significance as pathogens, these viruses, with their small DNA genomes and extensive reliance on the host system, are attractive model for the study of the host DNA replication and transcription (Huang et al, 2005). The begomovirus also receive much attention as vectors for the expression of foreign gene in plants. Considerable progresses have been made toward understanding begomovirus molecular biology and pathology, and recent reviews of this progress are available (Harrison & Robinson 1999). The begomovirus have a highly conserved replication system, whereas movement and other ancillary functions are quite divergent and appear to have originated from different sources. Given the ability of begomovirus DNA to undergo a variety of the rearrangements, it seems likely that these additional functions were acquired by recombination. Noticeably, the occurrence of recombinants is in high frequency (Fauquet et al., 2005; GarcÄ±´a-Andre´s et al., 2007), emerge often (Bananej et al., 2004; Monci et al., 2002; Padidam et al., 1995, 1999) and yield virulent strains that cause epidemics and molecular evolution (Zhou et al., 1997). It is crucial to find out the frequency with which the recombination occurs and the mechanistic and ecological features that control its rate. To achieve the predictability of emergence of new viruses will improve the control procedures which is solely dependent on the ability to identify the constraints to viral evolution (Fraile & García-Arenal, 2010). Evaluation of rate of recombination and determining the correlation with adaptability, virulence, and geographic distribution are important areas for future research. To explore the dynamics of nucleotide substitution in the begomoviruses from India, Pakistan, Nepal, Srilanka, China and Bangladesh, we undertook recombination detection and phylogenetic analysis of the retrieved sequences on the basis of highest recombination events.
Materials and Methods
The list of Begomovirus, were obtained from NCBI Taxonomy Browser and from that list only those species of Begomoviruses were selected those which were having cultivars from India, Bangladesh, China, Nepal, Pakistan and Sri Lanka (table 1.). The sequences of virus of Begomovirus family were retrieved from GenBank, which were obtained in FASTA format for further alignment. The sequences were analyzed by the multiple sequence alignment server Clustal-X 2 version which involves a progressive strategy for alignment of sequences. The basic information it provides is the identification of conserved sequence regions. This is very useful in designing experiments to test and modify the function and structure of nucleotides and in identifying new members of protein families.
Detection of Recombination events
For the full genome data set derived from GenBank in the FASTA format were analyzed through phylogenetic tools for identification of their ancestral relationships. Recombination event in Begomovirus were detected by using a software RDP2 i.e., Recombination Detection program. To further aid in evaluating evidence for recombination, RDP2 can also use phylip concurrently (Felsenstein, 1989; Olsen et al., 1994) to display phylogenetic trees. However, while the incidences of recombination infringe the postulation of coalescent analysis, this process is most likely to increase genetic variations and hence promote both the standard apparent substitution rate and its variability.
The aligned sequences were used for phylogenetic analysis by different methods like NJ, Maximun likelihood and Neighbourhood. The editing and visualization of tree were analysed by MEGA-4 (Tamura et al., 2007). The phylogenetic diversity analysis among selected sequences showed maximum recombination was done by split tree analysis (Rohayem et al. 2005). The order of sequence input is listed in table 2.
In this study, we sought to characterize recombination in Begomovirus reported from India and neighboring countries. Total 956 Begomovirus sequences were, down loaded from NCBI, out of which 437 viruses were from India, 367 from China, 115 from Pakistan, 20 from Bangladesh, 13 from Sri Lanka and 4 from Nepal (table 1). The viral strains used in this study were namely Jatropha mosaic India virus, Croton yellow vein mosaic virus, Indian cassava mosaic virus, Croton yellow vein mosaic virus, Cucumber mosaic virus, Jatropha mosaic India virus, Jatropha yellow mosaic India virus, Ageratum enation virus, Ageratum leaf curl virus, Ageratum yellow vein China virus, Ageratum yellow vein Sri Lanka virus, Bhendi yellow vein mosaic virus, Chilli leaf curl virus, Tomato leaf curl New Delhi virus, Cotton leaf curl Alabad virus, Cotton leaf curl Kokhran virus-Ganganagar, Cotton leaf curl Multan virus, Cotton leaf curl Rajasthan virus - India, Indian cassava mosaic virus, Sri Lankan cassava mosaic virus, Mungbean yellow mosaic India virus, Soybean yellow mosaic virus, Papaya leaf curl China virus, Pepper leaf curl virus, Squash leaf curl China virus, Sida yellow vein virus, Pumpkin yellow vein mosaic virus, Sweet potato leaf curl Bengal virus, Tobacco leaf curl Yunnan virus, Tomato leaf curl Bangalore virus, Tomato leaf curl China virus, Tomato leaf curl Gujarat virus, Tomato leaf curl Karnataka virus, Tomato leaf curl New Delhi virus, Tobacco curly shoot virus. The corresponding sequences of viruses were retrieved from Genbank, in FASTA format.
Recombination Detection in obtained sequences
Recombination events were evaluated by using a Recombination Detection Program (RDP version 2.0), this detected, the potential events, recombinant events in particular sequence, average P-value, ending breakpoints and beginning breakpoints, daughter as well as minor and major parent sequences. The analysis was performed with default settings for the detection method and a Bonferroni corrected P-value cut-off of 0.05. The Begomoviruses were grouped in 19 classes with 36 representatives, on the basis of the similar species and then recombination was detected between those sequences in those particular groups (table 3). The analysis has given results as shown in Table1. There were recombination found in most of the species but from all the recombinant sequences only those sequences were selected which has shown maximum number of recombination events. Hence, from the above analysis 36 sequences were identified with maximum number of recombination events, among them Tomato yellow leaf curl china virus was found to be 21039 potential events. The sequence gi|31744949|; gi|94960502|; gi|68051109|; gi|27805166| and gi|94960514| were found to be mother sequence. The and out of these 36 sequences it has been found that maximum number of potential events i.e., 21039 have been noticed in sequences which belong to species Tomato yellow leaf curl china virus and maximum number recombinant events detected were 1273. The other programe for detection of recombination have similar output at different bandwidth (Singh et al., 2008).
Phylogenetic analysis of recombinant sequences of Begomovirus
After obtaining the data for maximum recombination in Begomovirus species from India, Pakistan, China, Sri Lanka, Nepal and Bangladesh their phylogenetic analysis was carried out as shown in Figure 1. The phylogenetic tree was plotted on the basis of Maximum Likelihood among the maximum recombinant appeared sequences of Begomovirus from same species as well as from different species, hence total sequences were 36. The tree was visualized by TREE VIEW software. The analysis was done to detect phylogenetic evolution of the Begomovirus from the obtained data. Evidence for closely related phylogeny has been found among sequences derived from different species e.g. Ageratum enation virus accession no. gi|283468142|emb|AM701770.1| showed close relationship with Papaya leaf curl china virus accession no. gi|51507395|emb|AJ811914.1|, similarly Ageratum yellow vein virus accession no. gi|145834083|gb|EF527824.1| close relationship with Tomato leaf curl china virus accession no. gi|47155242|emb|AJ704618.1|, Squash leaf curl china virus accession no. gi|29825986|gb|AY184488.1| showed minimum distance with Tomato leaf curl Karnataka virus accession no. gi|57471334|gb|AY738101.1| and again Squash leaf curl china virus of another accession no. gi|283468151|emb|AM709505.1| showed close relation with Tomato leaf curl New Delhi virus accession no. gi|156767112|gb|EF620535.1| and Tomato leaf curl Bangalore virus accession no. gi|113196577|gb|DQ887537.1| showed least distance with Cotton leaf curl Multan virus accession no. gi|62528799|gb|AY765256.1| Hence from the above analysis inter-species as well as intra-species relations has been noticed among Begomovirus. To evaluate the diversity among the sequences shows the high recombination the split tree analysis was performed (figure 2). The split analysis for diversity have clumped in five major clusters with different isolates, the results are shown in table 4. We have got five major split among the representative sequences shown in table 4.
Detecting recombination is not easy because of its dependency on the amount of divergence between the sequences undergoing recombination, where recombination occurs along a sequence and how frequently, selection pressures, effective population size, mutation rate, and the methods used to measure recombination (Posada & Crandall 2001; Posada 2002). The Begomovirus bears circular ssDNA which replicates through double stranded intermediates by rolling circle mechanism (Saunders et al.1991; Stenger et al., 1991). An inspection of the large number of begomoviruses sequences currently available reveals that all the group members shares two features associated with the replication system. First, all begomoviruses genome contains an inverted repeat capable of forming hairpin (Sunter et al., 1985), and within the loop of the hairpin is the invariant sequence TAATATTAC. Second, all begomovirus genome encode an allele of the 40 kD AL1 (L1) protein, the only viral protein that is required for replication (Elmer et al., 1988). Despite an overall similarity in capsid and genome structure, the diversity among the members of the group is significant and needs to evaluate the rate and occurrence of the recombination. In general the recombination events are undetectable when exchange occurs between closely related sequences (Lazarowitz 1992 Rafaelo M. GalvaËœo,). In begomovirus the Homologous Intramolecular and Intermolecular recombination have been reported in some members are quite interesting. Begomovirus DNA also is subject to a variety of recombination mechanisms that do not involve homologous sequences. These illegitimate rearrangements may be inter- or intramolecular and nonhomologous recombination can generate subgenomic DNA found in begomovirus infected plants. Therefore to differentiate between recombination and mutation associated phenomenon among closely related sequences, is very difficult. These limitations confine the actual value of recombinant event which is always bigger than the recorded events (Posada et al., 2002; Hudson & Kaplan, 1985; Posada 20028). Therefore we have selected various isolates of Begomovirus on whole, reported from various parts of India, China, Pakistan, Srilanka, Bangladesh and Nepal, to detect the minute recombination also.
In several previous scenarios of Begomovirus emergence, it was suggested that indigenous viruses infecting weeds and wild hosts had been transferred to the new host, generating novel species after recombination and/or pseudorecombination events (Monci et al., 2002; Fraile & Garcia-Arenal, 2010; Paplomatas et al., 1994; Unseld et al., 1994; Pita et al., 2001; Saunders et al., 2002). Begomoviruses cause serious losses of many dicot crops throughout the warmer parts of the world, and intensive agriculture, as well as the global trade in agricultural products, has led to an ever-increasing significance of these viruses (Seal et al., 2006; Van Den Bosch et al., 2006). The severe economic consequences of emerging plant viruses highlight the importance of studies of plant virus evolution. One question of particular relevance is the extent to which the genomes of plant viruses are shaped by recombination (Chare & Hollmes, 2006). Recombination can provide selective advantage in the evolution of viruses within strains, species, genera and family (Keese & Gibbs 1993; Holland 1998). Recombination is very frequent in the evolution of Geminiviruses and occurs between species and within and across genera (Padidam et al., 1999). It has now been accepted that recombination contributed to the diversity of Geminiviruses and therefore, to the emergence of new variants and species reported worldwide. Interspecies recombination events have perhaps added considerably to the diversity of Begomoviruses and their appearance as pathogens of economic interest (Fondong et al., 2000). In the above study recombination has been detected in different species of Begomovirus especially focused on those species which were having cultivars from India, Pakistan, SriLanka, Bangladesh, Nepal and China. The sequences were analysed for the highest recombination events in particular sequences of the same species. 36 sequences had shown highest recombination events out of which Tomato yellow leaf curl china virus has rated the maximum number. The sequence was having the cultivar from China. Tomato leaf curl disease occurs in many tomato producing regions of the world including China, it is characterized by severe leaf curling, shrinking of tomato leaves and stunted plant growth. Studies have reported recombination in this virus species (Prassana & Rai 2007).
The phylogeny of Geminiviruses is extremely stable, and even by increasing the number of virus sequences from 36 in 1989 to 277 in 2002, the general structure of the tree has not changed, confirming the original concept of Howarth and Vandemark (1989) that it best represents this family of viruses. The geographical distribution of Geminiviruses, and Begomoviruses in particular, first noted by Harrison (1985), is still relevant today. Virus evolution is certainly an interesting and demanding topic, particularly as there is no fossil record available that can be used to probe early events. Phylogeny is the only tool currently available to explore the evolution of Geminiviruses, but their origin still remains a mystery.
The genus Begomovirus contains dicotyledonous infecting whitefly transmitted viruses in the family Geminiviridae. Begomoviruses are considered one of the largest and most successful groups of plant viruses that infect a wide range of crops, particularly in tropical and subtropical regions. They are responsible for numerous diseases of economically important crops, such as cassava, cotton, bean, pepper and tomato. A current consensus prediction for the extent of Begomovirus diversity holds that a high frequency of recombination resulted in the recent emergence of highly pathogenic virus genotypes causing a variety of serious Begomovirus diseases. Based on the "precision" of recombination events, DNA recombination can lead to various genetic changes. These include sequence insertions or duplications if the recombination endpoint in one of the recombining DNAs is upstream relative of the endpoint on the other DNA. In the study recombination was identified in most of the species of genus Begomovirus which were collected from India, Pakistan, SriLanka, Bangladesh, Nepal and China. The sequences were aligned to get the conserved sequences and then were gone through Recombination Detection Program, where maximum recombination events and potential events were taken into account. Then phylogenetic analyses of sequences showing maximum recombinant were done to investigate the viral evolution. The study performed by Padidam in 1999 supports the results derived from this work. Hence it could be concluded that phylogenetics-based sequence comparison and experimental approaches support the model that genetic recombination often causes changes in natural populations of plant viruses, resulting in enhanced pathogenecity, extended host range or overcoming host resistance factors. The emerging new virus genotype can compromise the effectiveness of antiviral strategies, underlying the importance of understanding viral DNA recombination. Given the importance of recombination in the molecular evolution of viruses, it is crucial to determine the frequency with which it occurs and the mechanistic and ecological features that control its rate (Hasiów-Jaroszewska et al., 2010). Being able to predict the emergence of new viruses and improve therapeutic procedures is dependent upon the ability to identify the constraints to viral evolution. Determining the relative rate of mutation in comparison to recombination and if they correlate with adaptability, virulence, and geographic distribution are important areas for future research.
The exchange of genetic material between different virus species, through recombination, has the potential to generate, unimaginable number of genetically distinct virus strains, including many that might cause deadly new plant diseases. Major fears with such a recombination induced strains of viruses are quick access to evolutionary innovations like broader host ranges, increased severities or altered tissue tropisms. Such study will be helpful for design of novel class virus resistant plant trough genetic engineering. While analyzing the sequences of Begomoviruses the chances of recombination are very high. Thus a unified approach is required to develop the new effective, virus resistance transgenic plant. Given the importance of recombination in the molecular evolution of viruses, it is crucial to determine the frequency with which it occurs and the mechanistic and ecological features that control its rate. Being able to predict the emergence of new viruses and improve strategies of plant protection is dependent upon the ability to categorize the constrictions to viral evolution. Therefore, an integrative approach of study is the present need to understand the pathogenic ability and host range.
Acknowledgement: Authors are thankful to Inha University, South Korea, and SHIATS Allahabad, India, for providing necessary environment.