Looking for the novel potato virus Y recombinant variants

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Abstract

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.

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About the authors

Aleksandr D. Antipov

All-Russian Research Institute of Agricultural Biotechnology

Email: antipovdm37@gmail.com
ORCID iD: 0000-0003-3522-1674
SPIN-code: 8244-7171
Russian Federation, 42 Timiryazevskaya st., Moscow, 127550

Yulia A. Surganova

All-Russian Research Institute of Agricultural Biotechnology

Email: yuliasyrganova@gmail.com
ORCID iD: 0009-0004-4148-9217
Russian Federation, 42 Timiryazevskaya st., Moscow, 127550

Vasiliy V. Taranov

All-Russian Research Institute of Agricultural Biotechnology

Email: v.taranov1@gmail.com
ORCID iD: 0000-0002-0728-0346
SPIN-code: 5008-4691
Scopus Author ID: 15761481700

Cand. Sci. (Biology)

Russian Federation, 42 Timiryazevskaya st., Moscow, 127550

Marina V. Lebedeva

All-Russian Research Institute of Agricultural Biotechnology

Author for correspondence.
Email: marilistik@mail.ru
ORCID iD: 0000-0001-5711-8331
SPIN-code: 1681-8890

Cand. Sci. (Biology)

Russian Federation, 42 Timiryazevskaya st., Moscow, 127550

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2. Fig. 1. Results of multiplex PCR for PVY diagnostics according to [15]. Sample numbering corresponds to Table 1. Marker — 50+ bp (Eurogen, Russia). The presence of 689 and 181 bp fragments indicates strains of the NO group, 452 and 181 bp fragments — strains of the NTN group.

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3. Fig. 2. Results of PVY genome amplification with Phusion polymerase (Thermo Scientific, USA) from cDNAs synthesized with different reverse transcriptases. 1 — Magnus (Eurogen, Russia), 2 — Maxima HMinus (Thermo Scientific, USA), 3 — HiScript III (Vazyme, China). Marker — 1 kb+ (Eurogen, Russia).

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4. Fig. 3. The most common recombinant variants between N and O strains with names according to [10]. Variants identified among the analyzed samples are highlighted in gray. Recombinant variants with atypical recombination points described for the first time are highlighted in green. The arrows indicate the annealing sites of the amplification primers.

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