Looking for the novel potato virus Y recombinant variants

BACKGROUND: Potato virus Y (PVY) is one of the most distributed plant RNA-viruses. PVY affects a wide host range including crops and wild plants from different families. Potato virus Y exists as a strain complex which produce a numerous recombinant variants. Usual diagnostic methods based on ELISA and PCR are able to define strain group at the best case, however not particular recombinant variant. Actually, only high-throughput sequencing of plant transcriptomes allow to obtain viral full-genome sequence and reveal all recombinant variants. However, the apply of this approach is limited by the relatively high cost of single sample analysis, which doesn’t allow to explore large-scale samples. AIM: The aim was to develop an approach for the bulk examination of PVY strain diversity. MATERIALS AND METHODS: Total RNA was isolated from potato leaves. cDNA was synthesized using different reverse transcriptases. PVY genome amplification was performed using different DNA polymerases. Amplicons were used for the sequencing libraries preparation, which were sequenced on MinION platform. Reads after processing were mapped on reference. SNP-calling was performed for the revealing different PVY isolates. RESULTS: The possibility of different reverse transcriptases and DNA polymerases for PVY genome amplification for farther sequencing was evaluated. Among exterminated sample 10 PVY isolates were revealed. The isolates belong to 5 different recombinant variants, three of them belong to common variants, while two another haven’t been previously described.One of them, N-Wi(s) is similar to the N-Wi, however the recombination point in Hc-Pro gene is shifted to the 5'-end and matches the NO-short variant. Another, SYR-IIa is similar to the SYR-II, however the recombination point in NIb gene is shifted about50 nucleotides to the 5'-end. CONCLUSION: Probably the diversity of PVY recombinant variants is still underestimated since even in the small exterminated sample two novel variants were found. Developed protocol allows to explore PVY strain diversity and has two main advantages: low cost of single sample and each read holds a whole viral genome allowing unambigously identify a recombinant variant.