Abstract
Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-022-00785-9.