In silico characterization of Tomato Leaf Curl New Delhi Virus in Manipur: Evidence for its global expansion and future threat

In silico characterization of Tomato Leaf Curl New Delhi Virus in Manipur: Evidence for its global expansion and future threat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In silico characterization of Tomato Leaf Curl New Delhi Virus in Manipur: Evidence for its global expansion and future threat Swati Chakraborty, Lourembam Sanajaoba Singh, Mritunjoy Barman, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2393085/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tomato leaf curl New Delhi virus (ToLCNDV) is a whitefly-transmitted bipartite ssDNA virus causing worldwide havoc to the tomato production. The present study addresses first report for the existence of ToLCNDV at Manipur region of North East India. Sequence comparison of DNA A (MG649330) and DNA B (OM752176) genomes showed close homogeneity of about 98% and 100% with Bangladesh isolates, but revealed only 84–96% (DNA A) and 81–100% (DNA B) similarity with other isolates of ToLCNDV. Phylogenetic relationship of global ToLCV species showed interchangeable results, substantiating that the isolate belonged to the old world bipartite group. Significant recombination events were ascertained around the intergenic region and the 5’end of the AC1 gene implicating a constant driving force for evolution and emergence of the strain. Population dynamics of global ToLCNDV isolates and other dominant ToLCV species within India were determined based upon several parameters i.e. genetic diversity, historic demographic events including neutrality test, Fst distance, Mismatch distribution plot, Haplotype network yielding significant results. Population genetics analysis overall showed low nucleotide diversity indicating recent population expansion. The dominant species of ToLCV in India also reinforced similar results where all the population showed diversifying selection constraint for the coat protein genes. Population dynamics of these viruses portrays Indian subcontinent as the possible hotspot for rapid demographic expansion from a small virus population size, indicating probability for rapid spread and emergence of distinct strains which may assist in future to signal the emergence of new threats to tomato production. Tomato Leaf Curl New Delhi Virus North East India Complete genome sequence Phylogeny Population Genetics Tomato Leaf Curl Disease Complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tomato ( Solanum lycopersicum Mill.) belonging to the family Solanaceae, is an extensively grown vegetable crop and is considered one of the most important "protective food" due to its special nutritive value. Several biotic and abiotic factors continue to ramify and cause considerable yield loss to this valuable crop. Among the biotic stress, Begomovirus genus under the family Geminiviridae is a major phytopathogen causing severe devastation in the tropical and sub-tropical regions. Tomato Leaf Curl Virus (TLCV) is a species of the Begomovirus which causes substantial loss to the tomato production worldwide and is considered one of the major begomovirus in India since its first outbreak. Besides this, emergence of novel insidious strains of these viruses along with the association of DNA satellites has been possible due to its efficient transmission by whitefly ( Bemisia tabaci Genn.) to a broad host range in a persistent, circulative and non-propagative manner. Prominent symptoms associated with this virus include reduced size, upward curling, and deformation with conspicuous yellow margins in the young leaves. Based on genomic organization and phylogenetic diversity, International Committee on Taxonomy of Viruses (ICTV) has classified the Geminiviridae family into nine genera embracing > 360 species such as Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Turncurtovirus and Topocuvirus (Brown et al., 2015 ; Zerbini et al., 2017 ). But recent Metagenomics research has led to the establishment of five new genera under the family Geminiviridae ( Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus , and Topilevirus ) to officially classify twelve new, divergent geminiviruses (Roumagnac et al., 2021). Based on genome characterization, geographical distribution and phylogenetic relationship, Begomovirus has been bifurcated into two major phylogenetic groups: Old World (OW: Europe, Africa, Indian subcontinent and Oceania) and New World (NW: America) begomoviruses (Briddon et al., 2006 and Rybicki, 1994 ). It has been long known that all begomoviruses (~ 140 species) native to the New World (NW) have bipartite genome having separate encapsidated DNA-A and DNA-B genome with a few reported exceptions (Macedo et al, 2018 ; Prabhandakavi, 2018; Fiallo-Olivé et al., 2020). Those originating from the Old World (OW) are majorly monopartite and comprises either only DNA-A, or DNA-A is occasionally associated with alpha or betasatellite molecules (Briddon and Stanley, 2006 ; Ferro et al., 2017 ; Ranjan et al., 2021 ). The virus genome size comprises of 2.6–2.8 kb and are encapsidated within a twinned icosahedral particle of size about 20×30 nm, assembled from 110 copies of a particular type of protein, coat protein (CP) (Briddon et al., 2003). In monopartite begomoviruses, the virion strand encodes V1 (coat protein) and V2 (pre-Coat protein) genes, the complementary virion strand (synthesized during the viral DNA replication) encode four genes viz. AC1 (replication associated protein), AC2 (transcription activator protein), AC3 (replication enhancing protein), and AC4 (Symptom enhancer protein) (Navot, 1991). An additional ORF AC5 may be present in some begomoviruses. Ilyas et al. ( 2010 ) reported that, ORF of AC5 is not present in all begomoviruses but has been found in DNA-A component of many begomoviruses. The virion sense strand of DNA B bears one gene BV1, whereas the complementary strand of DNA-B contains the BC1 gene involved in intra- and intercellular movement (Sanderfoot et al., 1996 ). In the past few years, there has been association of novel satellite DNA called betasatellites (formerly known as DNA-β) with mono and bi-partite begomoviruses to augment pathogenicity (Briddon et al. 2004 ). In addition, one smaller (approx. 1350 nucleotides) ssDNA molecule (termed alphasatellites) is also found in association with the Old World begomovirus-betasatellite complexes (Xie et al. 2010 ).Since the first report of ToLCD in India (Vasudeva and Samraj, 1948 ), a great diversity of begomoviruses has been reported in India, and many of these are still being regularly diagnosed and are in the process of genomic characterization. Majority of the begomoviruses infecting tomato in India contain monopartite genome except for two begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl Palampur virus (ToLCPalV) are bipartite in nature (Padidam et al., 1995 ; Kumar et al., 2008 ). Unlike other bipartite begomoviruses of the “New World”, ToLCNDV contains an AV2 gene in its DNA-A component. There has been lack of insight research about the whole genome characterization of ToLCV infecting tomato in the NE India. In such context the study of genome characteristics, distribution, and molecular variability of emerging begomovirus in India has become an eminent part of research nowadays. It has been vital to ascertain the genetic diversity within these viral species since these genetic variation directly affects the host-virus interactions which is eminent for viral evolution causing emergence of new strains and adaptation to new ecological niches (Sacristan and Garcia-Arenal, 2008; Gibbs et al., 2010 ). Nowadays genetic architecture and evolutionary analysis of virus populations has become a subject of increasing attention, hence our current study focuses on the whole genome characterization of the whole genome of ToLCV reported from Manipur in NE Indian province. Our study also aims to provide insights into ToLCNDV diversity and population structure at global level. Furthermore, our study also includes population dynamics of other dominant species of ToLCV in India with the aim to understand its evolutionary pattern. Hence the findings of this research could help to develop strategies to combat against the spread of these viruses and prevent formation of new strain development. Materials And Methods 2.1. Virus source, detection and cloning of full-length genome of the begomovirus A survey was conducted in the tomato growing areas of Manipur to ascertain the presence of tomato leaf curl virus in farmer’s field. The detection was based on the visual appearance of typical symptoms exhibited by begomovirus. Young symptomatic leaves or twigs of the infected plants from infected tomato field were collected in zipper plastic bags and brought into laboratory for molecular assay. Extraction of DNA from five samples was conducted by using genomic DNA isolation kit (Sigma-Aldrich, St Louis, USA). The concentration and purity of DNA samples was determined in a Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the eluted product was stored at − 80° for further use. The presence of the viral genome was preliminarily confirmed through polymerase chain reaction (PCR) using degenerate primer pairs (SPG1/SPG2) specific to Begomovirus (Li et al., 2018). Full- length genome of Tomato leaf curl virus was amplified by rolling circle amplification (RCA) with Φ29 DNA polymerase using TempliPhi TM DNA Amplification Kit (GE Healthcare, formerly Amersham). RCA products were then digested with Eco R1, pst1; MS1I, BamHI, XbaI, Sa1I , and restriction products were observed in a 1.5% agarose electrophoresis gel for RFLP analysis. One monomer band obtained by agarose gel purification, was ligated into a digested cloned into pUC18 vector to produce recombinant plasmids and transformed into competent Escherichia coli cells.(DH-5 alpha strain) Plasmids carrying DNA-A and DNA-B were bi-directionally sequenced by primer walking (Eurofins Genomics India Pvt. Ltd., Bangalore).The whole genome sequence was submitted to NCBI database for accession number. 2.2. Genomic Organisation and In silico analysis of Bipartite Begomovirus DNA-A genome was assembled with Unipro UGENE ver.1.12.2 at its default settings. Full-length genome sequence obtained by RCA and Sanger sequencing, were compared with the reported NCBI Database sequences by using CLUSTALW (MEGA 6) and pairwise sequence calculation was estimated using the Sequence Demarcation Tool (SDT) v.1.2. Best nucleotide substitution model with the aligned sequences was determined and Phylogenetic trees were constructed with 1000 iterations for bootstrapping by using MEGA 6 ver.6.0. The tree was rooted with an out-group and visualized using the Interactive Tree of Life (IOTL) (Letunic and Bork, 2007). The branch lengths of the tree were not proportional to the phylogenetic distance. Recombination Detection Program (RDP ver. 4) (Martin et al., 2015) was used for detection of potential recombination, identification of likely parental sequences, and localization of possible recombination breakpoints in multiple sequence alignments. Multiple sequence alignment was constructed using EBI Clustal W and the output was given as input for RDP ver. 4. To check the recombination breakpoints, the available methods such as RDP, BOOTSCAN, Chimaera, GENCOVE, SISSCAN, 3Seq, and MaxChi were used with their default settings (Martin et al., 2015). 2.4 Genetic diversity and haplotype network analysis To better understand the genetic structure, diversity and evolutionary pattern among the dominant species of ToLCV population within India and global ToLCNDV populations, several genetic parameters such as number of haplotypes (h), nucleotide diversity per site (π), mean haplotype diversity (Hd), average number of nucleotide differences (K), number of variable/segregating sites and genetic differentiation parameters among populations (Fst) were also examined using DnaSP ver. 6.12. Librado et al., 2009). In order to understand the relative contributions of various evolutionary pressure such as negative or purifying selection, neutrality, and diversifying or positive selection acting on the coat protein-coding gene, the ratio of nonsynonymous to synonymous substitution (dN/dS) was estimated using “estimate selection for each codon (Hyphy)” with Felsenstein,1981 model and maximum likelihood as statistical method in MEGA 10. Haplotype network was constructed using PopArt 1.7.based on the number and frequency of haplotypes in the virus population of ToLCNDV. The trait segment was incorporated into the nexus data file generated by DnaSP version 6.12.03 and was finally used as an input file for visualization based on the geographical source. 2.5 Population demography history In order to test for demographic changes based upon geographically separated populations, two approaches were followed Best fit demographic parameters for each population i.e. Tajima’s D (Tajima, 1989) and Fu-Li’s F (Fu, 1993) were applied using Arlequin 3.5 (Excoffier and Lischer, 2010) with p-values of 1,000 simulations to detect deviation from neutrality. Computation of the mismatch distributions by comparing the observed or actual frequency with the expected frequency of the population by calculating the Harpending’s raggedness index (r) (Harpending, 1994) with 1000 bootstrap approach in DnaSP version 6.12.03. The mismatch frequency graphs obtained from the software were plotted separately to determine whether the past populations size exhibited fluctuations or range expansion. In addition FST value which is a measure of pairwise genetic divergences between the populations was estimated with 10,000 permutations in Arlequin 3.5 (Excoffier and Lischer, 2010). The output file generated has been utilized in R software to depict the distribution graphically. Results 3.1. Detection and full length genome amplification PCR assays confirmed the begomovirus infection in all the five samples showing visual symptoms of typical ToLCV infection (Supplementary file1). The detection technique yielded expected band size of about 840bp. The full length sequences were annotated and submitted to NCBI Database after which the accession numbers MG649330 and OM752176 were obtained for DNA-A and DNA B component respectively. 3.2. Genome organization and in silico genome analysis 3.2.1 Genome organization and sequence analysis of DNA-A The BLAST analysis showed that the cloned nucleotide sequence exhibited the highest sequence identity (98.54%) with the DNA-A component of Jamalpur (ToLCNDV-[BD:Jam] KM383742) in the NCBI Database. Pairwise sequence comparison of DNA A genome of ToLCNDV Manipur isolate with 60 other ToLCV species of different country source revealed significant variation. ToLCNDV Manipur isolate revealed highest percent identity (98%) with KM383742_ToLCNDV_BD isolate but the nucleotide identity with other ToLCNDV isolates was between 89–93% i.e. less than 94% (Fig. 1 C). As per the conflict-resolution criteria for strain begomovirus taxonomy the Manipur isolate showed sequence similarity of less than 94% with other ToLCNDV isolates but its high similarity with the Bangladesh ToLCNDV isolate confirmed to not to be considered as a new strain. The DNA A component of ToLCNDV shared its highest similarity with the species ToLCPaV (83–85%) and showed 69–75% similarity with the other ToLCV species. Detailed nucleotide and amino acid sequence identities of individual genes of the bipartite genome shared between ToLCNDV Manipur isolate and those of other representative ToLCNDV isolates are provided in Table 1 . The assembled genome of DNA A constituted length of 2,740 basepair (Fig. 1 A), encoding seven ORFs, two (AV1 and AV2) in the virion sense and five (AC1, AC2, AC3, AC4 and AC5) in the complementary sense. The ORFs are separated by 274 nt long intergenic region (IR) that contains a non-nucleotide (TAATATTAC) sequence in a conserved a hairpin loop structure and incomplete direct repeats of an iteron [GGTGTA/C] adjacent to TATA box. 3.2.2 Genome organization and sequence analysis of DNA-B Similarly BLAST analysis of DNA-B genome showed Maximum similarity of 100% with DNA-B of AJ875158_ToLCNDV_BD showing little variation in the NCBI database as well as in SDT analysis. Pairwise sequence comparison through SDT analysis showed maximum differentiation (70%) with FJ660430_ToLCPaV_IR and showed about 81–100% with other ToLCNDV isolates (Fig. 1 D). The DNA-B genome is found to be more conserved than DNA-A genome. The assembled DNA-B genome constituted typical of New World begomoviruses with genome size 2688 base pair which comprises two ORFs i.e. BV1 and BC2 encoding movement protein and nuclear shuttle protein respectively (Fig. 1 B), Both the DNA-A and DNA-B genome constituted a typical origin of replication with inverted repeat sequences. Table1: Comparison of Nucleotide Identity (%) and Amino Acid Identity (%) of individual ORFs of DNA A and DNA B genome between ToLCNDV Manipur isolate with other ToLCNDV isolates. DNA-A Genome DNA-B Genome ToLCNDV Manipur isolate Other ToLCNDV isolates ToLCNDV Manipur isolate Other ToLCNDV isolates Genes Nucleotide Identity (%) Amino Acid Identity (%) Genes Nucleotide Identity (%) Amino Acid Identity (%) AV1 AV2 AC1 AC2 AC3 AC4 AC5 94.29-98.70 95.58-100 91-98.71 89.14-98.10 83.94-97.32 92.66-100 93.19-98.92 97.25-99.22 92.86-100 92.52-98.61 82.09-96.40 79.41-95.59 79.31-100 81.52-97.83 BV1 BC1 78-98 88-99 77-78 88-91 3.2.2 Phylogenetic Analysis The evolutionary relationship among 293 species of ToLCV of DNA A genome based upon geographical distribution was used in the study to understand the evolutionary pattern (Fig). The result obtained showed that all the ToLCV species were clustered according to their respective species under different clade for both the DNA and DNA B genome (Fig. 2 A). The DNA A genome of Manipur isolate (MG649330) shared its sister clade with the Bangladesh isolates (MT161674 and KM383742). Similarly the DNA B genome shared its sister clade with one of the Bangladesh isolate (AJ875158) (Fig. 2 B). Most of the ToLCV species were restricted according to their geographical origin except for the dominant species of ToLCV in India viz. ToLCKV, ToLCBV, ToLCPuV, ToLCGujV, ToLCJoyV, ToLCPaV,and ToLCNDV which were found to be clustered with the isolates of Pakistan and Bangladesh and weren’t limited to their geographical boundaries. Full forms of the acronyms along with the country source have been described in details in supplementary file 2. 3.2.3 Recombination Analyses Recombination analysis of DNA A anticipated the occurrence of a recombination event in the genome of ToLCNDV-Manipur isolate by seven methods programmed in the RDP4 software and out of which seven methods detected the positive results. SiScan was found with the lowest p value (p = 1.1480×10 25 ) and Maximum probability by RDP (9.055×10 − 04 ). Recombination breakpoints were detected at nucleotide positions 2570 (ORF C1) and 31 (intergenic region) of ToLCNDV, elucidating that this virus is recombinant nature. MK240306_ToLCNDV_Pakistan and MH577012_ToLCNDV_Gujarat were identified as putative major and minor parents respectively (Supplemetary file 1). Recombinant Putative Major Parent Putative Minor Parent Breakpoints a Methods b p-Value c Begin End MG649330_ToLCNDV_Manipur MK240306_ToLCNDV_Pakistan MH577012_ToLCNDV_Gujarat 2570 31 RBCST 9.055×10 − 04 3.3 Genetic diversity and Haplotype network analysis 3.3.1 Genetic diversity of Global ToLCNDV population 102 sequences were used for the above study revealing significant variable results. Several genetic parameters differentiating populations based upon country source i.e number of haplotypes (H), the haplotype diversity (h), and nucleotide diversity (p) for each population of ToLCNDV are presented in Table 2 . Out of 102 populations 97 haplotypes were formed where all the population showed high haplotype diversity (0.996 ± 0.002) and there was no shared haplotypes except for some Pakistan isolate strains, Indian isolate strains and Middle East population strains (Supplementary file 3). Haplotype diversity was quite high among all the populations but however on comparison the lowest was in the Pakistan population (0.89 ± 0.03). Nucleotide diversity was very low among all the populations (0.22 ± 0.00561) with the lowest value were in the populations of Mediterranean basin (0.001 ± 0.00) followed by populations of South-East Asian (0.10 ± 0.02) and Middle East populations (0.01 ± 0.02). Average number of nucleotide differences between sequences within population (K) showed similar results as the nucleotide diversity (493.54). The number of segregating (polymorphic) sites (S) among the populations showed different results (1709) where the highest value was observed in India (1443) followed by Pakistan (904), Bangladesh populations (617). The lowest value of S among the populations was found in Mediterranean basin (11) followed by Middle East (182) and South East Asian populations (182). Inter-population pairwise genetic distance (Fst) which is a measure of population differentiation between the six populations ranged from 0.18 to 0.95. Maximum differentiation was found between Mediterranean Basin and Middle East populations (0.83). The least differentiation was between India and Bangladesh populations (0.18) (Fig. 3 A). Table 2 Haplotype/nucleotide diversity, neutrality tests, and mismatch distribution values for all the ToLCNDV isolates based upon geographical distribution. Group Haplotype- nucleotide diversity Neutrality tests Demographic history POPULATION n h Hd ± SD π ± SD K S Tajima’sD Fu-Li’s F Harpending's Raggedness index(P) INDIA 48 46 1.00 0.19 410.61 1443 -0.26* -0.86* 0.0005 SRILANKA 1 1 - - - - - - - PAKISTAN 18 17 0.89 ± 0.03 0.13 ± 0.01 267.98 904 -0.17* -1.49** 0.0009 SOUTH EAST ASIA 11 11 1.00 ± 0.08 0.10 ± 0.02 21.92 182 -0.12 -0.98 0.0003 MIDDLE EAST 5 4 1.00 ± 0.08 0.01 ± 0.02 21.92 182 - - 0.0065 BANGLADESH 10 10 1.00 ± 0.004 0.17 ± 0.00 285.55 617 0.66** 0.67** 0.0586 MEDITERRANEAN BASIN 9 9 1.00 ± 0.008 0.001 ± 0.00 7.33 11 0.46 0.51 .07891 TOTAL POPULATION 102 97 0.996 ± 0.002 0.22 ± 0.005 493.54 1709 -0.10* -0.50* - n: Number of sequences, h: Number of Haplotypes, Hd: Haplotype (gene) diversity, π: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population, S: Number of variable/segregating sites.*, P < 0.05, **, P < 0.02. 3.3.2 Genetic Diversity of ToLCV species complex within India Eight distinct species of the genus Begomovirus (ToLCV) are highly prevalent in India and several genetic parameters were calculated within the species complex to understand the diversity pattern in India. The haplotype diversity was high and more or less similar among all the Begomovirus species (1.00 ± 0.01). The overall genetic diversity among all the species was very less (0.18 ± 0.07) with highest value was observed in ToLCJoyV (0.10 ± 0.07) population and lowest was observed in ToLCPatV (0.022 ± 0.05) population. The value of K was highest for ToLCJoyV (279.98) followed by ToLCNDV (205.36), ToLCBV (182.26), ToLCPaV (175.10), ToLCKV (142.21), ToLCPuV (139.01), and lowest value of K was for ToLCGujV (88.92). The value of S was highest for ToLCNDV (1489) and lowest for ToLCPuV (139) populations (Table 3 ). We also estimated the pattern of selective pressure on the CP by analyzing the dN/dS ratio. The estimates of dN/dS ratio for overall Indian populations was greater than 1 where some ToLCV species (Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur Virus) population within India showed the value of normalized rate of nonsynomous substitution (dN) greater than the rate of synonymous substitutions (dS) i.e. indication towards positive selection or diversifying selection. Rest of the populations of ToLCV species (Tomato leaf Curl Karnataka Virus, Tomato Leaf Curl Bangalore Virus, Tomato Leaf Curl Palampur Virus, and Tomato Leaf Curl Patna Virus) showed the value of dN/dS ratio close to 1 indicating resembling towards neutral evolution (Table 3 ). The Fst parameter within ToLCV species population in India showed maximum differentiation between ToLCPatV and ToLCGujV (0.91) and least differentiation was between ToLCNDV and ToLCGujV (0.58), indicating maximum gene flow. ToLCNDV had its maximum genetic distance with ToLCPatV (0.90) (Fig. 3 B). Table 3 Haplotype/nucleotide diversity, neutrality tests, and selection pressure values for all the dominant ToLCV species isolates in India based upon geographical distribution. Group Haplotype- nucleotide diversity Neutrality tests Selection Pressure POPULATION n h Hd ± SD π ± SD K S Tajima’sD Fu-Li’s F dN/dS Tomato Leaf Curl New Delhi Virus 48 46 1.000 ± 0.015 0.07536 ± 0.031 205.36 1489 -2.24163 -3.74018 1.78 Tomato leaf Curl Karnataka Virus 14 14 1.000 ± 0.027 0.05166 ± 0.006 142.21 493 -0.79059 -0.96677 0.750 Tomato Leaf Curl Bangalore Virus 21 21 1.000 ± 0.015 0.06761 ± 0.003 182.26 633 -0.54041 -0.82705 0.953 Tomato Leaf Curl Palampur Virus 20 20 1.000 ± 0.016 0.06407 ± 0.006 175.10 727 -1.09416 -1.74021 0.561 Tomato Leaf Curl Patna Virus 10 11 0.897 ± 0.067 0.02 ± 0.005 56.97 224 -1.13839 -0.75067 0.612 Tomato Leaf Curl Joydebpur Virus 26 26 1.000 ± 0.011 0.10 ± 0.070 279.98 1143 -1.21659 -0.92706 1.01 Tomato Leaf Curl Pune Virus 2 2 1.000 ± 0.500 0.05137 ± 0.025 139.01 139 - - - Tomato Leaf Curl Gujarat Virus 21 21 1.000 ± 0.015 0.03353 ± 0.008 88.92 632 -2.24580 -3.26502 1.60 TOTAL POPULATION 162 161 1.00 ± 0.01 0.18 ± 0.07 410.61 1443 -0.2680 -0.86 0.028 n: Number of sequences, h: Number of Haplotypes, Hd: Haplotype (gene) diversity, π: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population,S: Number of variable/segregating sites.*, P < 0.05, **, P < 0.02. 3.2 Haplotype network analysis In order to analyze the relationships between and among ToLCNDV population located globally, a haplotype network was constructed based upon DNA A. A dense network with 97 active haplotypes, some of which consisting of more than one sequence (2–3 sequences) was established with complicated relationship identified from 102 ToLCNDV whole genome sequences. ToLCV haplotypes present across the globe were found clustered according to their specific strain (Fig. 4 ) However the haplotypes of Mediterranean basin (H64-H72) and South East Asian (H73-H83) populations were more or less limited to their geographical boundaries except for the haplotypes of India, Bangladesh, Pakistan and Middle East populations which had higher prevalence and were found in diverse geographical locations globally. Two of the haplotype populations of Pakistan (H35) (MK240306_ToLCNDV_PK and MW426856_ToLCNDV_PK) shared its haplotype with one of the haplotype of Indian isolate (MT316389_ToLCNDV_Gujarat_India). Most of the Indian haplotypes (H1-H46) were found to be clustered with the haplotypes of Pakistan (H47-H62) and Bangladesh (H88-H97) population. Haplotypes of Middle East populations (H84-H87) were closely clustered with haplotypes of Indian populations (H12, H13, and H32). Details of the haplotypes of the ToLCNDV populations have been summoned in Supplementary file 3. 3.4 Population demography history To determine the occurrence and significance of any deviation from neutral evolution, Tajima’s D, and Fu and Li’s D and F values were calculated and overall the populations of ToLCNDV populations showed negative value of Tajima’s D (-0.10), and Fu and Li’s F (-0.50) values with significant p values except for the populations of Mediterranean basin (.46 to.51) suggesting population expansion. For demographic structure analysis of the geographically separated populations, examination of mismatch distribution of pairwise differences between sequences (Rogers and Harpending, 1992 ; Ray et al., 2003 ; Excoffier, 2005) was conducted (Fig. 5 ). The shape of the mismatch distribution plot for all populations except for the populations of Mediterranean basin showed unimodel and smooth shape justifying recent population expansion. The populations of Mediterranean basin suggest multimodel and ragged shape justifying stable or equilibrium population (Ray et al., 2003 ). Demographic parameters like Harpending’s raggedness index (Table 2 ) was calculated to ascertain whether an observed mismatch distribution is drawn from an expanded population or a stationary one, but none of the values for Harpending’s raggedness index were found significant which indicates that data shows coalescence to a model of population expansion (Harpending, 1994 ). Discussion There is a continuous emergence, spread and epidemic outbreak of ToLCD caused by different begomovirus species that collectively threatens tomato production worldwide. Previously there were reports of predominant occurrence of ToLCV strains in eastern and north eastern regions of India (Saha et al., 2014 ). Complete genome sequence of the virus has become imperative due to the growing number of species under this genus (Kings et al., 2011). Since there are very few convincing reports for the full genome characterization of the ToLCV species, hence this is the first comprehensive report for the evidence and characterization of ToLCNDV occurrence in Manipur, in North East Indian province. Whole genome amplification using RCA is still frequently used for the detection of several novel begomoviruses in tomato crops worldwide (Macedo et al., 2018 ; Medina et al., 2020 ; Agnihotri et al., 2018 ; Islam et al., 2019 ). Henceforth our present study includes isolation of complete genome of the begomovirus through RCA method, and as a result the virus exhibited the typical genome organization of the DNA-A and DNA-B component of OW bipartite begomovirus. Our homology based searches in the nucleotide databases and genome wide pairwise identity were consistent with the notion that the circular DNA virus was similar to ToLCNDV-Bangladesh which was earlier reported more than a decade ago in Bangladesh (Maruthi et al., 2007 ). Phylogenetic analysis based upon DNA A and DNA B also supported the fact that ToLCNDV-Manipur isolate belonged to the begomoviruses in the OW and closely related with other ToLCND begomovirus species. According to the guidelines of the International Committee on Taxonomy of Viruses (ICTV), DNA A genome of begomoviruses can be considered for taxonomic purposes, hence most of the analysis of the begomoviruses are conducted based upon genome DNA A (Brown et al., 2015 ). It has been well acquainted that recombination events at the intraspecific and interspecific genome levels are recurrent among begomoviruses, including tomato leaf curl New Delhi virus isolates (Fortes et al., 2016 ; Wilisiani et al., 2019). It has been well supported that recombination event drives the evolution of ToLCNDV (Moriones et al., 2017 ). Hence for this basis, recombination event was detected in ToLNDV-Manipur isolate along with other ToLCNDV iolates by seven different methods computed in the RDP4 program. Positively, the recombination event was detected around the intergenic region and the 5’end of the AC1 gene. Previously it has been reported that the N-terminal region of Rep and the adjacent intergenic region is a recombination hot-spot in the genome of begomoviruses (Lefeuvre et al., 2007 ; Lefeuvre et al., 2015). However, the results of our recombination analysis implies that ToLCNDV are distributed in a wide geographical areas from far North-east to far West regions which have been possible due to wide host adaptability and dissemination through whitefly to different ecological niches (Zhou et al., 1997 ) suggesting that recombination could play a significant role for development of new variants. Consequently, it may be suggested that ToLCNDV is under the constant drive for evolution and towards new strain development. Knowledge of the genetic diversity of the virus is of prime importance to discern the evolutionary pattern. Brown et al. in 2015 has mentioned earlier about the significance of genetic diversity for isolates of ToLCNDV. Keeping this in mind, we also determined the genetic variability among the global ToLCNDV populations based upon geographical locations in an attempt to determine the global genetic diversity of the species. In our present study we have investigated the genetic variation which revealed high haplotype diversity and low nucleotide diversity which can be a notion for a rapid demographic expansion from a small effective population size demarcated by geographical locations. Genetic diversity of the dominant ToLCV species in India was also investigated which also proved similar results supporting rapid spread and evolution of the ToLCV species complex. Haplotype network of ToLCNDV species based upon geographical locations demonstrates web like topology with a high ratio of singletons which is generally interpreted as indications of recent population expansion especially for the regions of Pakistan, India, and Bangladesh and to some extent for other countries such as Middle East. This is generally explicative for the fact that the population that has recently expanded in size from a small number of founders effect following a population bottleneck (Slatkin and Hudson, 1991 ). This demonstration is also supported by the results of neutrality test of Tajima’s D (Tajima, 1989 ) and Fu-Li’s F statistics (Fu and Li, 1993 ) which was carried out to evaluate the population history of the global ToLCNDV populations based upon geographical locations. Here, a negative value of Tajima’s D or Fu-Li’s F statistics signifies an excess of rare or low frequency polymorphism which relates to purifying selection, positive selection, or due to demographic history i.e. population expansion; in contrast a positive Tajima’s D or Fu-Li’s Fs statistics signifies low level of both high and low frequency polymorphism, signifying population subdivision event i.e. decline in population size and or balancing selection (Pichler, 2002 ). Estimates from Neutrality test of our study showed significant negative values for all the populations except for significant positive value for the populations of Mediterranean Basin. Earlier this has been well highlighted that the population of Mediterranean Basin is a discrete example of a founder effect associated with a population subdivision during the transmission to a new area, compromising adaptation to new environmental niches under selection pressure (Fraile et al., 1996 ; Monci et al., 2002 ; Fortes et al., 2016 ). One possible reason for population subdivision in ToLCNDV species in Mediterranean Basin regions was due to high genetic differentiation and low gene flow with other populations as well as low recombination with other ToLCNDV species which is well supported with genetic distance Fst data between the populations. Hence high gene flow between populations can slacken or restrict the process of geographic differentiation within each group that is reinforced by the result of Fst value. Notably, gene flow between the population of Indian isolates and Bangladesh isolates was least indicating chances of recombination for new strain. Our results for population expansion or subdivision are also well supported by the hypothesis that depicts whether observed data fit the sudden expansion model i.e. the raggedness index (Harpending, 1994 ). Our observations for the significant as well as non-significant value in goodness-of-fit distribution for all populations suggest that population expansion has occurred recently (Rogers, 1992 ). The population expansion among the dominant ToLCV species within Indian population also concomitantly fits with the above results. The possibility for this recent population expansion might be due to host diversification as well as robustness of the vector. For this reason there has been incessant report for ToLCV species and their variants in different geographical locations which may lead to new recombinants and novel strains of begomovirus (Heydarnejad et al., 2009 ; Kumar et al., 2008 ; Pratap et al., 2011 , Nagendran et al., 2016 ). Again it is well established that in order to detect positive selection in a population different genetics methods are required to analyze variations among sequences which includes (I) High proportion of non synonymous mutation. (II) Reduction in genetic diversity (III) High genetic differentiation between populations and (IV) High haplotype range (Hague and Routman, 2016 ). Traditionally, a protein is under neutral or normalized evolution when dN/dS = 1. Similarly, a protein is considered under the influence of positive selection when the rate of nonsynonymous substitutions (dN) is greater than the rate of synonymous substitutions (dS) i.e. >1. Conversely, protein is conserved i.e. under negative or purifying selection when the nonsynonymous substitutions (dN) is less than the rate of synonymous substitutions (dS) i.e. <1, hence harmful for fitness (Rodrigue, 2017 ). Our results fit collaterally with the interpretation of Hague and Routman, 2016 for the consideration of positive selection among the dominant ToLCV species viz. Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur Virus population in India. This may lead to the interpretation that Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur virus species are under the impact of recent population expansion and positive or diversifying selection. Plant pathogens undergo frequent selective pressures which changes with time that depends not only on the vagaries of the ecosystems but on the host diversity, robustness of whitefly as vector and incessant use of insecticides (Nigam, 2021 ). However, it was observed that low level of variability and negative or purifying selection among the isolates ToLCNDV in Italy (Mediterranean Basin) is probably due to recent introduction to the new area (Panno et al., 2019 ). Although closely-related ToLCNDV present predominantly in Indian Subcontinent, South East Asia, and Middle East Countries, is under constant population expansion and purifying selection, diverged group of the ToLCNDV has also been reported in Spain (ES) in Mediterranean Basin belonging to a different strain (Fortes et al., 2016 ). Since both the occurrence of monopartite and bipartite begomovirus in tomato is common in the Indian subcontinent, this mixed infection can lead to possible evolution and emergence of diverse strains in future (Seal et al., 2006 ; Matsuda et al., 2008 ; Moriones and Nvas-Castillo, 2008; Pita et al., 2001 ; Zhang and Zhou, 2010; Basu et al., 2021 ). Recently there has been report for more than 17 new begomovirus species in different countries (Quadros et al., 2019 ; Medina et al., 2020 ; Sohrab et al., 2020); hence there is chance for population expansion and emergence of novel strains of the virus. To conclude, this seminal work lays down the first wholesome report of bipartite begomovirus in NE Indian province, which suggests that in future more such reports can be possible leading to development of novel strains where recombination plays a major driving role for the evolution of ToLCNDV. In this present study our reappraisal was able to isolate and characterize the whole genome organization of ToLCNDV isolate in Manipur and determine it’s in silico properties. In addition the results of this study also contributed to determine the genetic diversity, identification and distribution of ToLCNDV in global tomato growing regions as well as among the dominant ToLCV species populations in India indicating future threat for its global population expansion. It is well known that population genetic diversity is greatly regulated by natural selection as well as random genetic drift, which in turn immensely depends upon several dynamic factors such as viral demography, virus genetic architecture, and viral ecology. Hence this type of investigation is of great potential in order to understand the epidemiology and develop more efficient control and treatment strategies against viral pathogens. Declarations Acknowledgments The first author thankfully acknowledges Bidhan Chandra Krishi Viswavidyalaya (ICAR accredited State Agricultural University) for allowing the research work. Author Contributions Swati Chakraborty: Conceptualization, Methodology, Software, Data analysis. Lourembam Sanajaoba Singh: Data curation, Conceptualization. Mritunjoy Barman: Data curation, Formal analysis, Writing-original draft preparation. Subham Dutta: Writing and Editing. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2393085","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":162401502,"identity":"45ee5ae1-ce33-4ab7-99ae-13786eb0d890","order_by":0,"name":"Swati Chakraborty","email":"","orcid":"","institution":"Bidhan Chandra Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Swati","middleName":"","lastName":"Chakraborty","suffix":""},{"id":162401505,"identity":"b3c96009-0437-4e71-a060-62b2d74c4d61","order_by":1,"name":"Lourembam Sanajaoba Singh","email":"","orcid":"","institution":"Bidhan Chandra Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lourembam","middleName":"Sanajaoba","lastName":"Singh","suffix":""},{"id":162401508,"identity":"aa69610d-3ca8-44a9-b73f-ad45500efa8e","order_by":2,"name":"Mritunjoy Barman","email":"","orcid":"","institution":"Bidhan Chandra Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mritunjoy","middleName":"","lastName":"Barman","suffix":""},{"id":162401509,"identity":"64f268c6-459f-4601-ab8e-677bd939cbcc","order_by":3,"name":"Subham Dutta","email":"","orcid":"","institution":"Bidhan Chandra Krishi Viswavidyalaya","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Subham","middleName":"","lastName":"Dutta","suffix":""},{"id":162401510,"identity":"2ba07d56-4f16-4379-9184-ed418f6085d4","order_by":4,"name":"Jayanta Tarafdar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACxgYILcc4g4GZgaECyGRmbiBCSwKDMTNYyxmQFkb8WiAggSGxHaSFsQ3ZZhyAuf2M4efCH3aJvbObHxvzzquN5m8HavlRsQ23w3pyjKVnJCQbz5xzzDiZd9vx3BmHGRsYe87cxuOXHANpngRm2Y0zcpgPztx2LLcBqIWZsQ2Plv43xr95EuoZ998AaZlzLHc+QS0zcsyAthxWbATakvCxoSZ3A2Etz8qsedKOGzMC/WLw4diB3I1ALQfx+cWwP3nzbR6bajlGYIhJJNTU5c47f/jggx8VeLQ0cBgg8w+DyQM41QOBPAP7A2R+HT7Fo2AUjIJRMEIBANiAXnoZ6K9rAAAAAElFTkSuQmCC","orcid":"","institution":"Bidhan Chandra Krishi Viswavidyalaya","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jayanta","middleName":"","lastName":"Tarafdar","suffix":""}],"badges":[],"createdAt":"2022-12-19 10:29:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2393085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2393085/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31046724,"identity":"e8891ce6-c2d0-453f-a6f2-f06dfc86cbfa","added_by":"auto","created_at":"2023-01-03 18:00:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333808,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome organizations of ToLCNDV-Manipur isolate infecting Tomato;\u003c/p\u003e\n\u003cp\u003eA: Schematic representation of genome position of DNA A and its ORFs. B: Schematic representation of genome position of DNA B and its ORFs. C: Pairwise homogeneity matrix based upon Nucleic Acid identity of DNA A genome among worldwide ToLCV species of different country source. D: Pairwise homogeneity matrix based upon Nucleic Acid identity of DNA B genome among worldwide ToLCV species of different country source.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/8eea35d7250f45aec289e31e.png"},{"id":31044615,"identity":"105c164e-6f27-4bfb-aa96-e37b4fdb6a12","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":389711,"visible":true,"origin":"","legend":"\u003cp\u003eA. Phylogenetic relationship based on nucleotide sequences of DNA A genome of worldwide ToLCV species on the basis of geographical distribution. B. Phylogenetic relationship based on nucleotide sequences of and DNA B genome of worldwide ToLCV species on the basis of geographical distribution.\u003c/p\u003e\n\u003cp\u003eThe branch lengths of the tree were not proportional to the phylogenetic distance and the bootstrap values were with 1000 replicates. The isolate under study is represented in bold form. The isolates from the same country are represented with the same colours as indicated in far right.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/cf49e1467b5d658c5b66b206.png"},{"id":31044620,"identity":"2c7a0786-1bf8-4699-b36e-fc86b8ff417b","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62214,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of Matrix of Pariwise Fst of worldwide ToLCNDV isolates based upon geographical distribution.\u003c/p\u003e\n\u003cp\u003eB. Diagrammatic representation of Matrix of Pariwise Fst of worldwide ToLCV species isolates in India based upon geographical distribution.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/348c2fd9c8dbf19fc345d0f2.png"},{"id":31046726,"identity":"ee2da499-21cf-42a6-84c4-e710ae58ea90","added_by":"auto","created_at":"2023-01-03 18:00:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":178645,"visible":true,"origin":"","legend":"\u003cp\u003eHaplotype network based on complete sequence of DNA A of worldwide ToLCNDV constructed using PopART (Leigh et al., 2015): median joining network of the worldwide ToLCNDV population based upon geographical distribution.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/ee82bb84cdb36b2b14e9b150.png"},{"id":31044619,"identity":"4f7af8aa-cea1-42a5-9271-769c9185b4cf","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176432,"visible":true,"origin":"","legend":"\u003cp\u003eMismatch distribution graphs ofworldwide ToLCNDV isolates according to geographical location of the ToLCNDVpopulation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/04b3751938207a32077399e2.png"},{"id":41642516,"identity":"26c6ffe7-25cb-4e1c-947f-e288a12d6443","added_by":"auto","created_at":"2023-08-16 14:37:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1531111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/2e9b705a-529e-497c-8f65-b2395097c00b.pdf"},{"id":31044618,"identity":"8bcaa119-61d0-48e0-a937-e6f828d15e04","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1287492,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/816d04c284b620eb67add530.pdf"},{"id":31046725,"identity":"cab32e01-a6b2-4d4b-a807-65bc98c9f651","added_by":"auto","created_at":"2023-01-03 18:00:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26825,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/87557fa905a6d178aa774116.xlsx"},{"id":31044622,"identity":"0893b3db-bacf-46d2-91f9-e137b6d3c6f8","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":735682,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/c47dc839f2a32b0d258644f6.pdf"},{"id":31044623,"identity":"778dbc76-64d6-4302-8cd9-a47bf5aa8eb8","added_by":"auto","created_at":"2023-01-03 17:52:01","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11743,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2393085/v1/8e88e1bb3e0d9024e1925504.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In silico characterization of Tomato Leaf Curl New Delhi Virus in Manipur: Evidence for its global expansion and future threat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e Mill.) belonging to the family Solanaceae, is an extensively grown vegetable crop and is considered one of the most important \"protective food\" due to its special nutritive value. Several biotic and abiotic factors continue to ramify and cause considerable yield loss to this valuable crop. Among the biotic stress, Begomovirus genus under the family \u003cem\u003eGeminiviridae\u003c/em\u003e is a major phytopathogen causing severe devastation in the tropical and sub-tropical regions. \u003cem\u003eTomato Leaf Curl Virus\u003c/em\u003e (TLCV) is a species of the Begomovirus which causes substantial loss to the tomato production worldwide and is considered one of the major begomovirus in India since its first outbreak. Besides this, emergence of novel insidious strains of these viruses along with the association of DNA satellites has been possible due to its efficient transmission by whitefly (\u003cem\u003eBemisia tabaci\u003c/em\u003e Genn.) to a broad host range in a persistent, circulative and non-propagative manner. Prominent symptoms associated with this virus include reduced size, upward curling, and deformation with conspicuous yellow margins in the young leaves.\u003c/p\u003e \u003cp\u003eBased on genomic organization and phylogenetic diversity, International Committee on Taxonomy of Viruses (ICTV) has classified the Geminiviridae family into nine genera embracing \u0026gt; 360 species such as Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Turncurtovirus and Topocuvirus (Brown et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zerbini et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). But recent Metagenomics research has led to the establishment of five new genera under the family \u003cem\u003eGeminiviridae\u003c/em\u003e (\u003cem\u003eCitlodavirus, Maldovirus, Mulcrilevirus, Opunvirus\u003c/em\u003e, and \u003cem\u003eTopilevirus\u003c/em\u003e) to officially classify twelve new, divergent geminiviruses (Roumagnac et al., 2021). Based on genome characterization, geographical distribution and phylogenetic relationship, Begomovirus has been bifurcated into two major phylogenetic groups: Old World (OW: Europe, Africa, Indian subcontinent and Oceania) and New World (NW: America) begomoviruses (Briddon et al., 2006 and Rybicki, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). It has been long known that all begomoviruses (~ 140 species) native to the New World (NW) have bipartite genome having separate encapsidated DNA-A and DNA-B genome with a few reported exceptions (Macedo et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Prabhandakavi, 2018; Fiallo-Olivé et al., 2020). Those originating from the Old World (OW) are majorly monopartite and comprises either only DNA-A, or DNA-A is occasionally associated with alpha or betasatellite molecules (Briddon and Stanley, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ferro et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ranjan et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The virus genome size comprises of 2.6–2.8 kb and are encapsidated within a twinned icosahedral particle of size about 20×30 nm, assembled from 110 copies of a particular type of protein, coat protein (CP) (Briddon et al., 2003).\u003c/p\u003e \u003cp\u003eIn monopartite begomoviruses, the virion strand encodes V1 (coat protein) and V2 (pre-Coat protein) genes, the complementary virion strand (synthesized during the viral DNA replication) encode four genes viz. AC1 (replication associated protein), AC2 (transcription activator protein), AC3 (replication enhancing protein), and AC4 (Symptom enhancer protein) (Navot, 1991). An additional ORF AC5 may be present in some begomoviruses. Ilyas et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) reported that, ORF of AC5 is not present in all begomoviruses but has been found in DNA-A component of many begomoviruses. The virion sense strand of DNA B bears one gene BV1, whereas the complementary strand of DNA-B contains the BC1 gene involved in intra- and intercellular movement (Sanderfoot et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). In the past few years, there has been association of novel satellite DNA called betasatellites (formerly known as DNA-β) with mono and bi-partite begomoviruses to augment pathogenicity (Briddon et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, one smaller (approx. 1350 nucleotides) ssDNA molecule (termed alphasatellites) is also found in association with the Old World begomovirus-betasatellite complexes (Xie et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).Since the first report of ToLCD in India (Vasudeva and Samraj, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1948\u003c/span\u003e), a great diversity of begomoviruses has been reported in India, and many of these are still being regularly diagnosed and are in the process of genomic characterization. Majority of the begomoviruses infecting tomato in India contain monopartite genome except for two begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl Palampur virus (ToLCPalV) are bipartite in nature (Padidam et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Unlike other bipartite begomoviruses of the “New World”, ToLCNDV contains an AV2 gene in its DNA-A component. There has been lack of insight research about the whole genome characterization of ToLCV infecting tomato in the NE India. In such context the study of genome characteristics, distribution, and molecular variability of emerging begomovirus in India has become an eminent part of research nowadays. It has been vital to ascertain the genetic diversity within these viral species since these genetic variation directly affects the host-virus interactions which is eminent for viral evolution causing emergence of new strains and adaptation to new ecological niches (Sacristan and Garcia-Arenal, 2008; Gibbs et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNowadays genetic architecture and evolutionary analysis of virus populations has become a subject of increasing attention, hence our current study focuses on the whole genome characterization of the whole genome of ToLCV reported from Manipur in NE Indian province. Our study also aims to provide insights into ToLCNDV diversity and population structure at global level. Furthermore, our study also includes population dynamics of other dominant species of ToLCV in India with the aim to understand its evolutionary pattern. Hence the findings of this research could help to develop strategies to combat against the spread of these viruses and prevent formation of new strain development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003e2.1. Virus source, detection and cloning of full-length genome of the begomovirus\u003c/h2\u003e\n\u003cp\u003eA survey was conducted in the tomato growing areas of Manipur to ascertain the presence of tomato leaf curl virus in farmer\u0026rsquo;s field. The detection was based on the visual appearance of typical symptoms exhibited by begomovirus. Young symptomatic leaves or twigs of the infected plants from infected tomato field were collected in zipper plastic bags and brought into laboratory for molecular assay. Extraction of DNA from five samples was conducted by using genomic DNA isolation kit (Sigma-Aldrich, St Louis, USA). The concentration and purity of DNA samples was determined in a Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the eluted product was stored at \u0026minus;\u0026thinsp;80\u0026deg; for further use. The presence of the viral genome was preliminarily confirmed through polymerase chain reaction (PCR) using degenerate primer pairs (SPG1/SPG2) specific to Begomovirus (Li et al., 2018). Full- length genome of Tomato leaf curl virus was amplified by rolling circle amplification (RCA) with \u0026Phi;29 DNA polymerase using TempliPhi TM DNA Amplification Kit (GE Healthcare, formerly Amersham). RCA products were then digested with \u003cem\u003eEco R1, pst1; MS1I, BamHI, XbaI, Sa1I\u003c/em\u003e, and restriction products were observed in a 1.5% agarose electrophoresis gel for RFLP analysis. One monomer band obtained by agarose gel purification, was ligated into a digested cloned into pUC18 vector to produce recombinant plasmids and transformed into competent Escherichia coli cells.(DH-5 alpha strain) Plasmids carrying DNA-A and DNA-B were bi-directionally sequenced by primer walking (Eurofins Genomics India Pvt. Ltd., Bangalore).The whole genome sequence was submitted to NCBI database for accession number.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.2. Genomic Organisation and\u003c/strong\u003e In silico \u003cstrong\u003eanalysis of Bipartite Begomovirus\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eDNA-A genome was assembled with Unipro UGENE ver.1.12.2 at its default settings. Full-length genome sequence obtained by RCA and Sanger sequencing, were compared with the reported NCBI Database sequences by using CLUSTALW (MEGA 6) and pairwise sequence calculation was estimated using the Sequence Demarcation Tool (SDT) v.1.2. Best nucleotide substitution model with the aligned sequences was determined and Phylogenetic trees were constructed with 1000 iterations for bootstrapping by using MEGA 6 ver.6.0. The tree was rooted with an out-group and visualized using the Interactive Tree of Life (IOTL) (Letunic and Bork, 2007). The branch lengths of the tree were not proportional to the phylogenetic distance. Recombination Detection Program (RDP ver. 4) (Martin et al., 2015) was used for detection of potential recombination, identification of likely parental sequences, and localization of possible recombination breakpoints in multiple sequence alignments. Multiple sequence alignment was constructed using EBI Clustal W and the output was given as input for RDP ver. 4. To check the recombination breakpoints, the available methods such as RDP, BOOTSCAN, Chimaera, GENCOVE, SISSCAN, 3Seq, and MaxChi were used with their default settings (Martin et al., 2015).\u003c/p\u003e\n\u003ch2\u003e2.4 Genetic diversity and haplotype network analysis\u003c/h2\u003e\n\u003cp\u003eTo better understand the genetic structure, diversity and evolutionary pattern among the dominant species of ToLCV population within India and global ToLCNDV populations, several genetic parameters such as number of haplotypes (h), nucleotide diversity per site (\u0026pi;), mean haplotype diversity (Hd), average number of nucleotide differences (K), number of variable/segregating sites and genetic differentiation parameters among populations (Fst) were also examined using DnaSP ver. 6.12. Librado et al., 2009). In order to understand the relative contributions of various evolutionary pressure such as negative or purifying selection, neutrality, and diversifying or positive selection acting on the coat protein-coding gene, the ratio of nonsynonymous to synonymous substitution (dN/dS) was estimated using \u0026ldquo;estimate selection for each codon (Hyphy)\u0026rdquo; with Felsenstein,1981 model and maximum likelihood as statistical method in MEGA 10. Haplotype network was constructed using PopArt 1.7.based on the number and frequency of haplotypes in the virus population of ToLCNDV. The trait segment was incorporated into the nexus data file generated by DnaSP version 6.12.03 and was finally used as an input file for visualization based on the geographical source.\u003c/p\u003e\n\u003ch2\u003e2.5 Population demography history\u003c/h2\u003e\n\u003cp\u003eIn order to test for demographic changes based upon geographically separated populations, two approaches were followed\u003c/p\u003e\n\u003col style=\"list-style-type: lower-roman;\"\u003e\n \u003cli\u003eBest fit demographic parameters for each population i.e. Tajima\u0026rsquo;s D (Tajima, 1989) and Fu-Li\u0026rsquo;s F (Fu, 1993) were applied using Arlequin 3.5 (Excoffier and Lischer, 2010) with p-values of 1,000 simulations to detect deviation from neutrality.\u003c/li\u003e\n \u003cli\u003eComputation of the mismatch distributions by comparing the observed or actual frequency with the expected frequency of the population by calculating the Harpending\u0026rsquo;s raggedness index (r) (Harpending, 1994) with 1000 bootstrap approach in DnaSP version 6.12.03. The mismatch frequency graphs obtained from the software were plotted separately to determine whether the past populations size exhibited fluctuations or range expansion. In addition FST value which is a measure of pairwise genetic divergences between the populations was estimated with 10,000 permutations in Arlequin 3.5 (Excoffier and Lischer, 2010). The output file generated has been utilized in R software to depict the distribution graphically.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Results","content":"\u003ch2\u003e3.1. Detection and full length genome amplification\u003c/h2\u003e\n\u003cp\u003ePCR assays confirmed the begomovirus infection in all the five samples showing visual symptoms of typical ToLCV infection (Supplementary file1). The detection technique yielded expected band size of about 840bp. The full length sequences were annotated and submitted to NCBI Database after which the accession numbers \u003cstrong\u003eMG649330\u003c/strong\u003e and \u003cstrong\u003eOM752176\u003c/strong\u003e were obtained for DNA-A and DNA B component respectively.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2. Genome organization and\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ein silico\u003c/span\u003e \u003cstrong\u003egenome analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2.1 Genome organization and sequence analysis of DNA-A\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe BLAST analysis showed that the cloned nucleotide sequence exhibited the highest sequence identity (98.54%) with the DNA-A component of Jamalpur (ToLCNDV-[BD:Jam] KM383742) in the NCBI Database. Pairwise sequence comparison of DNA A genome of ToLCNDV Manipur isolate with 60 other ToLCV species of different country source revealed significant variation. ToLCNDV Manipur isolate revealed highest percent identity (98%) with KM383742_ToLCNDV_BD isolate but the nucleotide identity with other ToLCNDV isolates was between 89\u0026ndash;93% i.e. less than 94% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). As per the conflict-resolution criteria for strain begomovirus taxonomy the Manipur isolate showed sequence similarity of less than 94% with other ToLCNDV isolates but its high similarity with the Bangladesh ToLCNDV isolate confirmed to not to be considered as a new strain. The DNA A component of ToLCNDV shared its highest similarity with the species ToLCPaV (83\u0026ndash;85%) and showed 69\u0026ndash;75% similarity with the other ToLCV species. Detailed nucleotide and amino acid sequence identities of individual genes of the bipartite genome shared between ToLCNDV Manipur isolate and those of other representative ToLCNDV isolates are provided in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe assembled genome of DNA A constituted length of 2,740 basepair (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), encoding seven ORFs, two (AV1 and AV2) in the virion sense and five (AC1, AC2, AC3, AC4 and AC5) in the complementary sense. The ORFs are separated by 274 nt long intergenic region (IR) that contains a non-nucleotide (TAATATTAC) sequence in a conserved a hairpin loop structure and incomplete direct repeats of an iteron [GGTGTA/C] adjacent to TATA box.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2.2 Genome organization and sequence analysis of DNA-B\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSimilarly BLAST analysis of DNA-B genome showed Maximum similarity of 100% with DNA-B of AJ875158_ToLCNDV_BD showing little variation in the NCBI database as well as in SDT analysis. Pairwise sequence comparison through SDT analysis showed maximum differentiation (70%) with FJ660430_ToLCPaV_IR and showed about 81\u0026ndash;100% with other ToLCNDV isolates (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The DNA-B genome is found to be more conserved than DNA-A genome. The assembled DNA-B genome constituted typical of New World begomoviruses with genome size 2688 base pair which comprises two ORFs i.e. BV1 and BC2 encoding movement protein and nuclear shuttle protein respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), Both the DNA-A and DNA-B genome constituted a typical origin of replication with inverted repeat sequences.\u003c/p\u003e\n\u003cp\u003eTable1: Comparison of Nucleotide Identity (%) and Amino Acid Identity (%) of individual ORFs of DNA A and DNA B genome between ToLCNDV Manipur isolate with other ToLCNDV isolates.\u003c/p\u003e\n\u003ctable align=\"left\" border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"1015\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"52.01970443349754%\"\u003e\n \u003cp\u003eDNA-A Genome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"47.98029556650246%\"\u003e\n \u003cp\u003eDNA-B Genome\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.04433497536946%\"\u003e\n \u003cp\u003eToLCNDV Manipur isolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"34.97536945812808%\"\u003e\n \u003cp\u003eOther ToLCNDV isolates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.019704433497537%\"\u003e\n \u003cp\u003eToLCNDV Manipur isolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"35.960591133004925%\"\u003e\n \u003cp\u003eOther ToLCNDV isolates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.04433497536946%\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.24137931034483%\"\u003e\n \u003cp\u003eNucleotide Identity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.733990147783253%\"\u003e\n \u003cp\u003eAmino Acid Identity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.019704433497537%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Genes \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.748768472906406%\"\u003e\n \u003cp\u003eNucleotide Identity (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.211822660098523%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Amino Acid Identity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.04433497536946%\"\u003e\n \u003cp\u003eAV1\u003c/p\u003e\n \u003cp\u003eAV2\u003c/p\u003e\n \u003cp\u003eAC1\u003c/p\u003e\n \u003cp\u003eAC2\u003c/p\u003e\n \u003cp\u003eAC3\u003c/p\u003e\n \u003cp\u003eAC4\u003c/p\u003e\n \u003cp\u003eAC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.24137931034483%\"\u003e\n \u003cp\u003e94.29-98.70\u003c/p\u003e\n \u003cp\u003e95.58-100\u003c/p\u003e\n \u003cp\u003e91-98.71\u003c/p\u003e\n \u003cp\u003e89.14-98.10\u003c/p\u003e\n \u003cp\u003e83.94-97.32\u003c/p\u003e\n \u003cp\u003e92.66-100\u003c/p\u003e\n \u003cp\u003e93.19-98.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.733990147783253%\"\u003e\n \u003cp\u003e97.25-99.22\u003c/p\u003e\n \u003cp\u003e92.86-100\u003c/p\u003e\n \u003cp\u003e92.52-98.61\u003c/p\u003e\n \u003cp\u003e82.09-96.40\u003c/p\u003e\n \u003cp\u003e79.41-95.59\u003c/p\u003e\n \u003cp\u003e79.31-100\u003c/p\u003e\n \u003cp\u003e81.52-97.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.019704433497537%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBV1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.748768472906406%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e78-98\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e88-99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.211822660098523%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e77-78\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e88-91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e3.2.2 Phylogenetic Analysis\u003c/h2\u003e\n\u003cp\u003eThe evolutionary relationship among 293 species of ToLCV of DNA A genome based upon geographical distribution was used in the study to understand the evolutionary pattern (Fig). The result obtained showed that all the ToLCV species were clustered according to their respective species under different clade for both the DNA and DNA B genome (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The DNA A genome of Manipur isolate (MG649330) shared its sister clade with the Bangladesh isolates (MT161674 and KM383742). Similarly the DNA B genome shared its sister clade with one of the Bangladesh isolate (AJ875158) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Most of the ToLCV species were restricted according to their geographical origin except for the dominant species of ToLCV in India viz. ToLCKV, ToLCBV, ToLCPuV, ToLCGujV, ToLCJoyV, ToLCPaV,and ToLCNDV which were found to be clustered with the isolates of Pakistan and Bangladesh and weren\u0026rsquo;t limited to their geographical boundaries. Full forms of the acronyms along with the country source have been described in details in supplementary file 2.\u003c/p\u003e\n\u003ch2\u003e3.2.3 Recombination Analyses\u003c/h2\u003e\n\u003cp\u003eRecombination analysis of DNA A anticipated the occurrence of a recombination event in the genome of ToLCNDV-Manipur isolate by seven methods programmed in the RDP4 software and out of which seven methods detected the positive results. SiScan was found with the lowest p value (p\u0026thinsp;=\u0026thinsp;1.1480\u0026times;10\u003csup\u003e25\u003c/sup\u003e) and Maximum probability by RDP (9.055\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;04\u003c/sup\u003e). Recombination breakpoints were detected at nucleotide positions 2570 (ORF C1) and 31 (intergenic region) of ToLCNDV, elucidating that this virus is recombinant nature. MK240306_ToLCNDV_Pakistan and MH577012_ToLCNDV_Gujarat were identified as putative major and minor parents respectively (Supplemetary file 1).\u003c/p\u003e\n\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003ctable border=\"1\" id=\"Taba\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRecombinant\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePutative Major Parent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePutative Minor Parent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eBreakpoints\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMethods\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ep-Value c\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBegin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMG649330_ToLCNDV_Manipur\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMK240306_ToLCNDV_Pakistan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMH577012_ToLCNDV_Gujarat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRBCST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.055\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;04\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch2\u003e3.3 Genetic diversity and Haplotype network analysis\u003c/h2\u003e\n\u003ch2\u003e3.3.1 Genetic diversity of Global ToLCNDV population\u003c/h2\u003e\n\u003cp\u003e102 sequences were used for the above study revealing significant variable results. Several genetic parameters differentiating populations based upon country source i.e number of haplotypes (H), the haplotype diversity (h), and nucleotide diversity (p) for each population of ToLCNDV are presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Out of 102 populations 97 haplotypes were formed where all the population showed high haplotype diversity (0.996\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002) and there was no shared haplotypes except for some Pakistan isolate strains, Indian isolate strains and Middle East population strains (Supplementary file 3). Haplotype diversity was quite high among all the populations but however on comparison the lowest was in the Pakistan population (0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03). Nucleotide diversity was very low among all the populations (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00561) with the lowest value were in the populations of Mediterranean basin (0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00) followed by populations of South-East Asian (0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) and Middle East populations (0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02). Average number of nucleotide differences between sequences within population (K) showed similar results as the nucleotide diversity (493.54). The number of segregating (polymorphic) sites (S) among the populations showed different results (1709) where the highest value was observed in India (1443) followed by Pakistan (904), Bangladesh populations (617). The lowest value of S among the populations was found in Mediterranean basin (11) followed by Middle East (182) and South East Asian populations (182).\u003c/p\u003e\n\u003cp\u003eInter-population pairwise genetic distance (Fst) which is a measure of population differentiation between the six populations ranged from 0.18 to 0.95. Maximum differentiation was found between Mediterranean Basin and Middle East populations (0.83). The least differentiation was between India and Bangladesh populations (0.18) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHaplotype/nucleotide diversity, neutrality tests, and mismatch distribution values for all the ToLCNDV isolates based upon geographical distribution.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eHaplotype- nucleotide diversity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNeutrality tests\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDemographic history\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePOPULATION\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHd\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026pi;\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTajima\u0026rsquo;sD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFu-Li\u0026rsquo;s F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHarpending\u0026apos;s Raggedness index(P)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINDIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e410.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1443\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.26*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.86*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRILANKA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePAKISTAN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e267.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.17*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.49**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSOUTH EAST ASIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMIDDLE EAST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0065\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBANGLADESH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e285.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.66**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMEDITERRANEAN BASIN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.07891\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTOTAL POPULATION\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e102\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.996\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e493.54\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1709\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.10*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.50*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003en: Number of sequences, h: Number of Haplotypes, Hd: Haplotype (gene) diversity, \u0026pi;: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population, S: Number of variable/segregating sites.*, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **, P\u0026thinsp;\u0026lt;\u0026thinsp;0.02.\u003c/p\u003e\n\u003ch2\u003e3.3.2 Genetic Diversity of ToLCV species complex within India\u003c/h2\u003e\n\u003cp\u003eEight distinct species of the genus Begomovirus (ToLCV) are highly prevalent in India and several genetic parameters were calculated within the species complex to understand the diversity pattern in India. The haplotype diversity was high and more or less similar among all the Begomovirus species (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01). The overall genetic diversity among all the species was very less (0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) with highest value was observed in ToLCJoyV (0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) population and lowest was observed in ToLCPatV (0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) population. The value of K was highest for ToLCJoyV (279.98) followed by ToLCNDV (205.36), ToLCBV (182.26), ToLCPaV (175.10), ToLCKV (142.21), ToLCPuV (139.01), and lowest value of K was for ToLCGujV (88.92). The value of S was highest for ToLCNDV (1489) and lowest for ToLCPuV (139) populations (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eWe also estimated the pattern of selective pressure on the CP by analyzing the dN/dS ratio. The estimates of dN/dS ratio for overall Indian populations was greater than 1 where some ToLCV species (Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur Virus) population within India showed the value of normalized rate of nonsynomous substitution (dN) greater than the rate of synonymous substitutions (dS) i.e. indication towards positive selection or diversifying selection. Rest of the populations of ToLCV species (Tomato leaf Curl Karnataka Virus, Tomato Leaf Curl Bangalore Virus, Tomato Leaf Curl Palampur Virus, and Tomato Leaf Curl Patna Virus) showed the value of dN/dS ratio close to 1 indicating resembling towards neutral evolution (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe Fst parameter within ToLCV species population in India showed maximum differentiation between ToLCPatV and ToLCGujV (0.91) and least differentiation was between ToLCNDV and ToLCGujV (0.58), indicating maximum gene flow. ToLCNDV had its maximum genetic distance with ToLCPatV (0.90) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHaplotype/nucleotide diversity, neutrality tests, and selection pressure values for all the dominant ToLCV species isolates in India based upon geographical distribution.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eHaplotype- nucleotide diversity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNeutrality tests\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSelection Pressure\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePOPULATION\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHd\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026pi;\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTajima\u0026rsquo;sD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFu-Li\u0026rsquo;s F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003edN/dS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl New Delhi Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07536\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e205.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.24163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.74018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato leaf Curl Karnataka Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05166\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.79059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.96677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.750\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Bangalore Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06761\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.54041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.82705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.953\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Palampur Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06407\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.09416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.74021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Patna Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.897\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.13839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.75067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.612\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Joydebpur Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e279.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.21659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.92706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Pune Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato Leaf Curl Gujarat Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03353\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.24580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.26502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTOTAL POPULATION\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e162\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e161\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e410.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1443\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.2680\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003en: Number of sequences, h: Number of Haplotypes, Hd: Haplotype (gene) diversity, \u0026pi;: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population,S: Number of variable/segregating sites.*, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **, P\u0026thinsp;\u0026lt;\u0026thinsp;0.02.\u003c/p\u003e\n\u003ch2\u003e3.2 Haplotype network analysis\u003c/h2\u003e\n\u003cp\u003eIn order to analyze the relationships between and among ToLCNDV population located globally, a haplotype network was constructed based upon DNA A. A dense network with 97 active haplotypes, some of which consisting of more than one sequence (2\u0026ndash;3 sequences) was established with complicated relationship identified from 102 ToLCNDV whole genome sequences. ToLCV haplotypes present across the globe were found clustered according to their specific strain (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) However the haplotypes of Mediterranean basin (H64-H72) and South East Asian (H73-H83) populations were more or less limited to their geographical boundaries except for the haplotypes of India, Bangladesh, Pakistan and Middle East populations which had higher prevalence and were found in diverse geographical locations globally. Two of the haplotype populations of Pakistan (H35) (MK240306_ToLCNDV_PK and MW426856_ToLCNDV_PK) shared its haplotype with one of the haplotype of Indian isolate (MT316389_ToLCNDV_Gujarat_India). Most of the Indian haplotypes (H1-H46) were found to be clustered with the haplotypes of Pakistan (H47-H62) and Bangladesh (H88-H97) population. Haplotypes of Middle East populations (H84-H87) were closely clustered with haplotypes of Indian populations (H12, H13, and H32). Details of the haplotypes of the ToLCNDV populations have been summoned in Supplementary file 3.\u003c/p\u003e\n\u003ch2\u003e3.4 Population demography history\u003c/h2\u003e\n\u003cp\u003eTo determine the occurrence and significance of any deviation from neutral evolution, Tajima\u0026rsquo;s D, and Fu and Li\u0026rsquo;s D and F values were calculated and overall the populations of ToLCNDV populations showed negative value of Tajima\u0026rsquo;s D (-0.10), and Fu and Li\u0026rsquo;s F (-0.50) values with significant p values except for the populations of Mediterranean basin (.46 to.51) suggesting population expansion.\u003c/p\u003e\n\u003cp\u003eFor demographic structure analysis of the geographically separated populations, examination of mismatch distribution of pairwise differences between sequences (Rogers and Harpending, \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e; Ray et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e; Excoffier, 2005) was conducted (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The shape of the mismatch distribution plot for all populations except for the populations of Mediterranean basin showed unimodel and smooth shape justifying recent population expansion. The populations of Mediterranean basin suggest multimodel and ragged shape justifying stable or equilibrium population (Ray et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). Demographic parameters like Harpending\u0026rsquo;s raggedness index (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) was calculated to ascertain whether an observed mismatch distribution is drawn from an expanded population or a stationary one, but none of the values for Harpending\u0026rsquo;s raggedness index were found significant which indicates that data shows coalescence to a model of population expansion (Harpending, \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is a continuous emergence, spread and epidemic outbreak of ToLCD caused by different begomovirus species that collectively threatens tomato production worldwide. Previously there were reports of predominant occurrence of ToLCV strains in eastern and north eastern regions of India (Saha et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Complete genome sequence of the virus has become imperative due to the growing number of species under this genus (Kings et al., 2011). Since there are very few convincing reports for the full genome characterization of the ToLCV species, hence this is the first comprehensive report for the evidence and characterization of ToLCNDV occurrence in Manipur, in North East Indian province.\u003c/p\u003e\u003cp\u003eWhole genome amplification using RCA is still frequently used for the detection of several novel begomoviruses in tomato crops worldwide (Macedo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Medina et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Agnihotri et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Islam et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Henceforth our present study includes isolation of complete genome of the begomovirus through RCA method, and as a result the virus exhibited the typical genome organization of the DNA-A and DNA-B component of OW bipartite begomovirus. Our homology based searches in the nucleotide databases and genome wide pairwise identity were consistent with the notion that the circular DNA virus was similar to ToLCNDV-Bangladesh which was earlier reported more than a decade ago in Bangladesh (Maruthi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Phylogenetic analysis based upon DNA A and DNA B also supported the fact that ToLCNDV-Manipur isolate belonged to the begomoviruses in the OW and closely related with other ToLCND begomovirus species. According to the guidelines of the International Committee on Taxonomy of Viruses (ICTV), DNA A genome of begomoviruses can be considered for taxonomic purposes, hence most of the analysis of the begomoviruses are conducted based upon genome DNA A (Brown et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It has been well acquainted that recombination events at the intraspecific and interspecific genome levels are recurrent among begomoviruses, including tomato leaf curl New Delhi virus isolates (Fortes et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wilisiani et al., 2019). It has been well supported that recombination event drives the evolution of ToLCNDV (Moriones et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Hence for this basis, recombination event was detected in ToLNDV-Manipur isolate along with other ToLCNDV iolates by seven different methods computed in the RDP4 program. Positively, the recombination event was detected around the intergenic region and the 5’end of the AC1 gene. Previously it has been reported that the N-terminal region of Rep and the adjacent intergenic region is a recombination hot-spot in the genome of begomoviruses (Lefeuvre et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lefeuvre et al., 2015). However, the results of our recombination analysis implies that ToLCNDV are distributed in a wide geographical areas from far North-east to far West regions which have been possible due to wide host adaptability and dissemination through whitefly to different ecological niches (Zhou et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) suggesting that recombination could play a significant role for development of new variants. Consequently, it may be suggested that ToLCNDV is under the constant drive for evolution and towards new strain development.\u003c/p\u003e\u003cp\u003eKnowledge of the genetic diversity of the virus is of prime importance to discern the evolutionary pattern. Brown et al. in 2015 has mentioned earlier about the significance of genetic diversity for isolates of ToLCNDV. Keeping this in mind, we also determined the genetic variability among the global ToLCNDV populations based upon geographical locations in an attempt to determine the global genetic diversity of the species. In our present study we have investigated the genetic variation which revealed high haplotype diversity and low nucleotide diversity which can be a notion for a rapid demographic expansion from a small effective population size demarcated by geographical locations. Genetic diversity of the dominant ToLCV species in India was also investigated which also proved similar results supporting rapid spread and evolution of the ToLCV species complex. Haplotype network of ToLCNDV species based upon geographical locations demonstrates web like topology with a high ratio of singletons which is generally interpreted as indications of recent population expansion especially for the regions of Pakistan, India, and Bangladesh and to some extent for other countries such as Middle East. This is generally explicative for the fact that the population that has recently expanded in size from a small number of founders effect following a population bottleneck (Slatkin and Hudson, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). This demonstration is also supported by the results of neutrality test of Tajima’s D (Tajima, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and Fu-Li’s F statistics (Fu and Li, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) which was carried out to evaluate the population history of the global ToLCNDV populations based upon geographical locations. Here, a negative value of Tajima’s D or Fu-Li’s F statistics signifies an excess of rare or low frequency polymorphism which relates to purifying selection, positive selection, or due to demographic history i.e. population expansion; in contrast a positive Tajima’s D or Fu-Li’s Fs statistics signifies low level of both high and low frequency polymorphism, signifying population subdivision event i.e. decline in population size and or balancing selection (Pichler, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Estimates from Neutrality test of our study showed significant negative values for all the populations except for significant positive value for the populations of Mediterranean Basin. Earlier this has been well highlighted that the population of Mediterranean Basin is a discrete example of a founder effect associated with a population subdivision during the transmission to a new area, compromising adaptation to new environmental niches under selection pressure (Fraile et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Monci et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Fortes et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). One possible reason for population subdivision in ToLCNDV species in Mediterranean Basin regions was due to high genetic differentiation and low gene flow with other populations as well as low recombination with other ToLCNDV species which is well supported with genetic distance Fst data between the populations. Hence high gene flow between populations can slacken or restrict the process of geographic differentiation within each group that is reinforced by the result of Fst value. Notably, gene flow between the population of Indian isolates and Bangladesh isolates was least indicating chances of recombination for new strain. Our results for population expansion or subdivision are also well supported by the hypothesis that depicts whether observed data fit the sudden expansion model i.e. the raggedness index (Harpending, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Our observations for the significant as well as non-significant value in goodness-of-fit distribution for all populations suggest that population expansion has occurred recently (Rogers, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The population expansion among the dominant ToLCV species within Indian population also concomitantly fits with the above results. The possibility for this recent population expansion might be due to host diversification as well as robustness of the vector. For this reason there has been incessant report for ToLCV species and their variants in different geographical locations which may lead to new recombinants and novel strains of begomovirus (Heydarnejad et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pratap et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Nagendran et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAgain it is well established that in order to detect positive selection in a population different genetics methods are required to analyze variations among sequences which includes (I) High proportion of non synonymous mutation. (II) Reduction in genetic diversity (III) High genetic differentiation between populations and (IV) High haplotype range (Hague and Routman, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Traditionally, a protein is under neutral or normalized evolution when dN/dS = 1. Similarly, a protein is considered under the influence of positive selection when the rate of nonsynonymous substitutions (dN) is greater than the rate of synonymous substitutions (dS) i.e. \u0026gt;1. Conversely, protein is conserved i.e. under negative or purifying selection when the nonsynonymous substitutions (dN) is less than the rate of synonymous substitutions (dS) i.e. \u0026lt;1, hence harmful for fitness (Rodrigue, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our results fit collaterally with the interpretation of Hague and Routman, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e for the consideration of positive selection among the dominant ToLCV species viz. Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur Virus population in India. This may lead to the interpretation that Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Gujarat Virus, and Tomato Leaf Curl Joydebpur virus species are under the impact of recent population expansion and positive or diversifying selection.\u003c/p\u003e\u003cp\u003ePlant pathogens undergo frequent selective pressures which changes with time that depends not only on the vagaries of the ecosystems but on the host diversity, robustness of whitefly as vector and incessant use of insecticides (Nigam, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, it was observed that low level of variability and negative or purifying selection among the isolates ToLCNDV in Italy (Mediterranean Basin) is probably due to recent introduction to the new area (Panno et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although closely-related ToLCNDV present predominantly in Indian Subcontinent, South East Asia, and Middle East Countries, is under constant population expansion and purifying selection, diverged group of the ToLCNDV has also been reported in Spain (ES) in Mediterranean Basin belonging to a different strain (Fortes et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Since both the occurrence of monopartite and bipartite begomovirus in tomato is common in the Indian subcontinent, this mixed infection can lead to possible evolution and emergence of diverse strains in future (Seal et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Matsuda et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Moriones and Nvas-Castillo, 2008; Pita et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Zhang and Zhou, 2010; Basu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently there has been report for more than 17 new begomovirus species in different countries (Quadros et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Medina et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sohrab et al., 2020); hence there is chance for population expansion and emergence of novel strains of the virus.\u003c/p\u003e\u003cp\u003eTo conclude, this seminal work lays down the first wholesome report of bipartite begomovirus in NE Indian province, which suggests that in future more such reports can be possible leading to development of novel strains where recombination plays a major driving role for the evolution of ToLCNDV. In this present study our reappraisal was able to isolate and characterize the whole genome organization of ToLCNDV isolate in Manipur and determine it’s in silico properties. In addition the results of this study also contributed to determine the genetic diversity, identification and distribution of ToLCNDV in global tomato growing regions as well as among the dominant ToLCV species populations in India indicating future threat for its global population expansion. It is well known that population genetic diversity is greatly regulated by natural selection as well as random genetic drift, which in turn immensely depends upon several dynamic factors such as viral demography, virus genetic architecture, and viral ecology. Hence this type of investigation is of great potential in order to understand the epidemiology and develop more efficient control and treatment strategies against viral pathogens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first author thankfully acknowledges Bidhan Chandra Krishi Viswavidyalaya (ICAR accredited State Agricultural University) for allowing the research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSwati Chakraborty: Conceptualization, Methodology, Software, Data analysis. Lourembam Sanajaoba Singh: Data curation, Conceptualization. Mritunjoy Barman: Data curation, Formal analysis, Writing-original draft preparation. Subham Dutta: Writing and Editing. Jayanta Tarafdar: Review, Validation, Visualization, Investigation, Funding Acquisition, Project administration, Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance of ethical standard\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest regarding the publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgnihotri, A.K., Mishra, S.P., Tripathi, R.C., Ansar, M., Srivastava, A., \u0026amp; Tripathi, I.P. (2018). 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Evidence that DNA-A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination. \u003cem\u003eJournal of General Virology, \u003c/em\u003e\u003cem\u003e78\u003c/em\u003e, 2101\u0026ndash;2111. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tomato Leaf Curl New Delhi Virus, North East India, Complete genome sequence, Phylogeny, Population Genetics, Tomato Leaf Curl Disease Complex","lastPublishedDoi":"10.21203/rs.3.rs-2393085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2393085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTomato leaf curl New Delhi virus (ToLCNDV) is a whitefly-transmitted bipartite ssDNA virus causing worldwide havoc to the tomato production. The present study addresses first report for the existence of ToLCNDV at Manipur region of North East India. Sequence comparison of DNA A (MG649330) and DNA B (OM752176) genomes showed close homogeneity of about 98% and 100% with Bangladesh isolates, but revealed only 84\u0026ndash;96% (DNA A) and 81\u0026ndash;100% (DNA B) similarity with other isolates of ToLCNDV. Phylogenetic relationship of global ToLCV species showed interchangeable results, substantiating that the isolate belonged to the old world bipartite group. Significant recombination events were ascertained around the intergenic region and the 5\u0026rsquo;end of the AC1 gene implicating a constant driving force for evolution and emergence of the strain. Population dynamics of global ToLCNDV isolates and other dominant ToLCV species within India were determined based upon several parameters i.e. genetic diversity, historic demographic events including neutrality test, Fst distance, Mismatch distribution plot, Haplotype network yielding significant results. Population genetics analysis overall showed low nucleotide diversity indicating recent population expansion. The dominant species of ToLCV in India also reinforced similar results where all the population showed diversifying selection constraint for the coat protein genes. Population dynamics of these viruses portrays Indian subcontinent as the possible hotspot for rapid demographic expansion from a small virus population size, indicating probability for rapid spread and emergence of distinct strains which may assist in future to signal the emergence of new threats to tomato production.\u003c/p\u003e","manuscriptTitle":"In silico characterization of Tomato Leaf Curl New Delhi Virus in Manipur: Evidence for its global expansion and future threat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-03 17:51:55","doi":"10.21203/rs.3.rs-2393085/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8f7e3f9-db28-4535-9b79-103363ce530b","owner":[],"postedDate":"January 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-16T14:29:21+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-03 17:51:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2393085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2393085","identity":"rs-2393085","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}