Next-generation sequencing reveals genetic variation in ToCV infecting Pakistani tomato plants

Tomato chlorosis virus (ToCV), causing yellowing disease in tomato, is a phloem-limited, whiteflytransmitted crinivirus that mainly affects plants by reducing chlorophyll content.During a survey conducted in 2019, cirinivirus-specific yellowing symptoms were observed in the tomato fields in Multan, Khanewal, and Lodhran districts of Pakistan.To confirm the etiology of the yellowing disease of tomato, a total of 76 symptomatic and 22 asymptomatic samples were collected out of which 72% of the tested plants were found to be positive for ToCV using RT-PCR.The full genomic nucleotide sequences of two selected ToCVisolates were obtained by next-generation sequencing (NGS).RNA1 and RNA2 of each isolate comprised 8594 and 8242 nucleotides, respectively.The NGS results were verified by sequencing the amplified overlapping fragments of RNA1 and RNA2 using specific primers designed in this study.In BLASTn analysis for ToCV-Pak1 and ToCV-Pak2, RNA1 from both isolates had the highest similarities (99.41-99.46%)with a South Korean isolate; RNA2 had the highest similarities of 99.67-99.64%with a Greek isolate for Pak1 and Turkish isolate for Pak2, respectively.Phylogenetic analysis indicated that RNA1 of the Pakistani isolates clustered together, forming a subclade, and RNA2 from one of the Pakistani isolates (MN869006) clustered with a Brazilian isolate.At the same time, the other one (MN869007) fell close to a Turkish isolate.Statistical analysis indicated a low polymorphic frequency in RNA1 but a higher polymorphic frequency in RNA 2 quantifying the degree of variation in the analyzed isolates which may