Identification and Partial Genome Characterization of Mungbean Yellow Mosaic India Virus Infecting Soybean via Rolling Circle Amplification and Restriction-based Cloning

Abstract

Background: Yellow mosaic disease (YMD) is one of the most economically damaging constraints on soybean (Glycine max L.) cultivation across South and Southeast Asia. It is caused by whitefly (Bemisia tabaci)-transmitted, bipartite begomoviruses belonging to the family Geminiviridae. In the present study, a soybean plant showing typical bright yellow mosaic symptoms was investigated to identify and molecularly characterise the causal begomovirus. Methods: Total genomic DNA extracted from symptomatic leaf tissue was subjected to rolling circle amplification (RCA) using bacteriophage phi29 DNA polymerase. The resulting high-molecular-weight concatemeric product was digested with four restriction endonucleases (HindIII, EcoRI, NotI and BamHI). The HindIII fragment, which produced the sharpest and most intense band, was cloned into the HindIII site of dephosphorylated pUC18 vector. Recombinant clones were screened by colony PCR using component-specific primers. Plasmid DNA from positive clones was subjected to double digestion with HindIII and BglI to confirm full-length insertion, followed by sequencing of the cloned insert. The resulting sequence was analysed using BLASTn against the NCBI nucleotide database, and viral identity was assigned using pairwise BLAST identity comparisons. Results: Each restriction enzyme released a single, unit-length fragment of approximately 2.7 kb, consistent with a monopartite circular ssDNA genome component. Colony PCR amplified the expected ~508 bp diagnostic product, confirming successful cloning. Double digestion with HindIII and BglI released the ~2.7 kb insert from the 1.5 kb and 1.2 kb vector backbone fragments, confirming full-length insertion. Sequencing yielded a partial genome sequence of ~2.5 kb, which showed 96–99% nucleotide sequence identity with DNA-A components of Mungbean yellow mosaic India virus (MYMIV) isolates reported from multiple geographic regions of India. As the recovered sequence represented a partial rather than a complete DNA-A component, phylogenetic reconstruction was not performed. Conclusion: These results confirm that MYMIV, rather than the closely related Mungbean yellow mosaic virus (MYMV), is associated with yellow mosaic disease in the soybean accession examined. The study demonstrates a straightforward RCA–RFLP cloning pipeline for recovering full-length begomoviral genome components directly from field-infected tissue without prior knowledge of the infecting strain.