Micro-evolution of Beet necrotic yellow vien virus during a single crop season using next-generation sequencing

Galein, Yann [UCL] Champeil, Agnès [ITB] Escriou, Hervé [ITB] Richard-Molard, Marc [ITB] Bragard, Claude [UCL]

In order to understand the impact of the sugar beet variety on the evolution of the Beet necrotic yellow vein virus, 454 deep-sequencing was used to generate sequences from BNYVV RNA-2, RNA-3 and RNA-5 targeted amplicons. The samples were collected in field plots where the disease distribution had been characterized, with mostly presence of the BNYVV type P associated with the tetrad SYHG and the fifth genomic RNA. Trial plots were selected from a long term experiment conduced jointly with the French technical institute in the historic BNYVV epidemic epicenter, in Yèvre-la-Ville, Loiret, France, from 2008 to 2012. Three different varieties were used with different BNYVV resistance background susceptible (Rz1rz1rz2rz2), tolerant (Rz1rz1rz2rz2) and resistant (Rz1rz1Rz2rz2). The estimated disease root impact in September varied from 2% in the “resistant” variety, 6% in the tolerant and 22% in the susceptible one. Amplicons were obtained using tagged primers positioned in conserved BNYVV genome region. The genes targeted were anticipated to be involved in the interaction plant virus : the p25 of BNYVV RNA3, the p14 of BNYVV RNA2 and the p26 of BNYVV RNA5 to create a library composed of nine cDNA samples. All cDNA were then pooled together for emulsion PCR. The nine different tags allowed to retain the link to the original sample, the gene and the variety. The 454 deep-sequences were analyzed all samples. Approximately 6000-7000 sequences per sample for each amplicon/ variety combination were obtained. The analysis of conventional sequences obtained early and at the end of the sugar beet growing season, together with sequences from bioassays using a susceptible (rz1rz1rz2rz2) variety as well as deep sequencing results reveals variations according to the sugar beet genotype as well as to the spatial dispersion of the virus.