The determination by single read sequencing of the amino acid tetrad motif at position 67 to 70 of the p25 protein coded by RNA3 is a common tool to identify the highly virulent resistance breaking isolates. Recent studies have shown however that other sequence motifs at various RNA species could play a role on the virulence of the isolate. The whole genome deep-sequencing of the different RNA species is therefore the appropriate technique to reveal

the maximum of genetic diversity and its potential impact on virulence and plant resistance.

The objective of this study was to use an alternative protocol to produce cDNA’s of all RNA species of a number of BNYVV isolates and compare the generated genetic variability to the standard single read sequencing method.

Materials and Methodsi. BnYVV isolatesIn total 12 Rhizomania infected samples have been analyzed originating either from infested soil or infected plants. One sample coded as ‘63-Xavier virus 2012’ represents a purified BNYVV sample kindly provided by Prof. Dr. David Gilmer (IBMP laboratory; Strasbourg). The soil samples were taken from commercial sugarbeet fields at the different locations as shown in table I. Infected root samples were generated by sowing a Rhizomania susceptible genotype as bait plant. The 5 infected plant samples from the different geographical origins all represent infected lateral root samples harvested on Rhizomania susceptible genotypes. Prior to RNA extraction, a TAS-ELISA was performed on all samples according to standard procedures to confirm the presence of BNYVV.

ii. nucleic acid extract, cdna amplicon library and deep-sequencingTotal RNA was extracted from an homogenized root from a single plant using the RNeasy plant mini kit (Qiagen, Germany). Preparation and amplification of double-stranded cDNA libraries from total RNA were done using the Ovation® RNA-Seq System V1 (PART NO. 7100, Nugen Technologies, Inc., CA, USA) with random primers. The cDNA libraries were sent off to GATC for whole genome sequencing. The Roche GS FLX+ System (Titanium XL+ sequencing chemistry) was applied on the pooling of the 12 tagged libraries producing 25.000 to 40.000 reads in total with a typically reached mode read length of 550 to 650 bases up to 1000 bp.

iii. Bioinformatic analysisAll sequence data were analyzed using the CLC Main Workbench software using the following workflow: 1) Groom Fastq files; 2) Generate quality report; 3) Trim reads on quality (default), length (< 25 bp), ambiguous nucleotides (default); 4) Remove duplicate reads; 5) Map the reads to the sugar beet genome assembly and collect the unmapped reads; 6) Run de novo assembly on the unmapped reads; 7) Blast the contigs > 500 bp on the BNYVV reference genome of NCBI (RNA1, NC_003514; RNA2, NC_003515; RNA3, NC_003516; RNA4, NC_003517; RNA5, NC_003513); 8) Identification of the SNPs; 9) Perform a Blastx of the unmapped contigs on the translated nucleotide NCBI database.

iV. PCr virus detectionThe presence of the viruses BNYVV, BVQ (Beet virus Q) and BSBV (Beet soil-borne virus) and the vector Polymyxa betae (P. betae) were analyzed and tested by a multiplex RT-PCR detection (Meunier et al., 2003). The presence of BCV2 (Beet cryptic virus 2) was analyzed through a RT-PCR with in house designed primers. Regarding the virus LBVaV (Lettuce big-vein associated virus), its presence was analyzed with the primers described by Alemzadeh et al. 2012. Finally, for the detection of Olpidium brassicae, the multiplex of J. A. Herrera-Vasquez et al. (2009) was used.

resultsi. nucleotide variation rna3 & 5: single read vs. whole genome sequencing

The table 2 summarizes the single-nucleotide polymorphism (SNP’s) for the p25 and p26 ORF generated by single read sequencing. The two major differences for p25 type discrimination are indicated in red and green, respectively non synonymous mutations which discriminate between the BNYVV pathotypes and synonymous mutations discriminating between pathotypes. Besides the tetrad zone, 11 positions were detected inducing non-synonymous mutations.

Regarding the p26, only 1 discriminative synonymous mutation was detected to distinguish between the P-type and the J-type. In the P-type, an additional synonymous mutation was detected at the position 92.

ii. Whole genome deep-sequencingsnP variation: The number of SNP’s within 500 bp lengths in comparison to the NCBI reference sequence for all RNA species is shown in figure 1. Overall the genetic variation for RNA1 and RNA2 is the highest among the RNA species except for the Flevo isolate where no SNP’s could be detected at any of the RNA’s. Regarding the variation at RNA3 & 5, only few mutations were detected for RNA3 while RNA5 contained much more variation depending on the nucleotide position.

Fig. 2: Proportion of viral contigs detection within the cDNA pool of 12 isolates.

detection of viral sequences: Blasting of the amplified sequences of all 12 isolates onto NCBI gene bank also showed a large proportion of the viral contigs other than BNYVV sequences. The proportion of non BNYVV sequences is summarized in figure 2. BSBV and BVQ both vectored by P. betae represent nearly 25% of the viral sequences. The remaining sequences (~10%) could be assigned to LBVaV and BCV2, the latter being vectored by Olpidium species. The presence of both viruses including the Olpidium sp. was reconfirmed by PCR specific amplification (figure 3 to 5).

Fig. 4: PCR amplified DNA of BCV2. Fig. 5 – PCR amplified DNA of LBVaV. Fig. 6 – A. Multiplex PCR amplified DNA of Olpidium spp. (977 bp for O. bornovanus / 579 bp for O. virulentus / 204 bp for O. brassicae -triple infection detection) (Herrera-Vásquez et al., 2009). B. Multiplex PCR amplified DNA of Olpidium spp. in this study.

Lane 1, 11-Seewinckel; 2, 25-Rougemont 2003; 3, 27-California 2004; 4, 29-Gatinais-sol Cherokee; 5, 3-Artenay; 6, 31-Gatinais-Rhizo forte; 7, 35-Rougemont 2010; 8, 36-Illkoffen 2004; 9, 47-Damiel 2009; 10, 63-Xavier virus 2012; 11, 64-Bondaroy; 12, 37-Flevo; T+, positive control; Bl, blanko (sterile water); M1, Gene Ruler 100bp DNA Ladder; M2, Gene Ruler 1kb DNA Ladder (Promega Corporation, USA).

w w w . s e s v a n d e r h a v e . c o m

Genetic diversity of the Bnyvv virus By whole Genome sequencinG – some new insiGhtsde Bruyne e.1, willems G.1, Broos L.1, Galein Y.1, hermes J.2 1 SESVanderHave N.V., Industriepark Soldatenplein Zone 2 Nr 15, 3300 Tienen, Belgium tel. +32 (0)16 808 2112 Université catholique de Louvain, Faculté des Sciences, Place des Science B-1348 Louvain-la-Neuve, Belgique tel. +32 (0)10 47 33 24

table 1: Overview of Rhizomania isolates used for whole genome deep-sequencing (WGS).

Fig. 1: Number of SNP’s expressed as % relative to the NCBI reference sequences for 12 BNYVV isolates.

The figure 3 summarizes the nucleotide variation detected for the full lengths of all RNA species. Although this data set cannot be compared to the single read sequencing data because different lengths of RNA 3 & 5 were amplified, it is clear that the number of SNP/mutations detected for the RNA5 is much higher using the WGS approach compared to the single read sequencing using RNA3 specific primers for the ORF of p25. Regarding RNA3, difference in generated variation between both approaches is less obvious.

Fig. 3: Nucleotide variation for full length RNA species of 12 BNYVV isolates.

ConClusions and disCussion• TheuseoftheOvation®RNA-SeqSystemV1

wassuccessfullyappliedtotheBNYVVisolatesasanapproachtoperformwholegenomedeepsequencing.TheresultsonthedetectedgeneticvariabilityatRNA5whenpresentwasparticularinterestinginthewaythatmuchmorevariationcouldbedetectedcomparedtosinglereadsequencing.Whetherthisvariationisoutsidethep26ORFremainsunclearandneedstobeconfirmedinanotherstudy.HoweveritcanbestatedthattheprocedureusedtogeneratecDNAfragmentsofanyoftheRNAspeciescanimpactthefinalresultregardinggeneticvariability.

• TheuseoftheovationkitmoreparticularlytheuseofrandomprimersforthecDNAproductionenabledustohavemoreinsightinthepresenceofnonBNYVVviralsequences.BesidesthecommonviralfloraassociatedwithBNYVVsuchasBVQ,BSBVtwonewvirustypeswereclearlydetectedandreconfirmedbyspeciesspecificPCRamplification.TheroleofthesevirusesinrelationtoBNYVVremainsunclearbutpotentialinteractionwithimplicationsattheplantlevelcannotbeexcluded.ThepresenceoftheLettucebig-vein associated virusvectoredbyOlpidiumspp.needsfurtherstudytoprovetheintercellularpresenceofthevectorandthisvirusinsugarbeetrootcells.TheroleofBeet cryptic virus 2whichistransmittedbysugarbeetpollenalsoneedsfurtherstudy.table 2: Mutation position for the 12 samples in single read sequencing.