| dc.description.abstractEn | INTRODUCTIONIn population genetics, repeated DNA sequences are widely targetted to describe genetic diversity. In bacteriology, Variable Numbers of Tandem Repeats (VNTR) are widely used as such repeats do not vary at the same pace as do neutral gene genes (Hood et al., 1996; Frotingham and Meeker-O'Connell, 1998) and usually evolve in a recA-independent manner (Puopolo et al., 2001). When used in combinaison (Multiple Loci VNTR Analysis, MLVA) (Johanss0n et al., 2004), they produce fingerprints of bacterial populations, especially at the site of emergence which translate as a bottle-neck in terms of genetic variability. In the frame of a “Candidatus Phytoplasma solani” (CaPsol) genome survey of the PO strain, a gene, named Coll-like, was found to encode GXY amino acid repeats reminiscent of collagen structure (Cimerman et al., 2006). Search for other GXY repeats in CaPsol strain PO draft genome revealed the presence GXY repeats in a gene encoding a surface protein. The encoded protein is also possessing large repeated domains upstream of the collagen-like repeats, a signal peptide and a C-terminal hydrophobic alpha-helix, a structure reminiscent of Vmp1 (Cimerman et al., 2009). This gene will be referred as vmp3-coll. The objective a this study was to assess the variability of this two potential VNTR in grapevine Bois noir-associated CaPsol isolates.MATERIALS AND METHODS Total nucleic acids were extracted from 1 g of grapevine petioles according to CTAB standard procedure from samples collected in Bekaa valley - Lebanon and from various location in Croatia. This samples had been shown to be positive for CaPsol infection (Mortada et al., 2013; Plavec et al., 2015). They were submitted to nested-PCR amplification with the primers described below. For coll-like, the first primer pair was collF1 5’-CCTTTTATCATAACCTGTTT-3’ collR1 CAATAGGATATAGATAG followed by the primer pair collF2 (5’-GCTATTATTTATGCTGGCTC-3’) / collR2 (5’-CGCTGTCCGTCTTTTGCTAA-3’) and PCR conditions were 95°C 2 min, 35 cycles of 95°C 30 sec, 55°C 30 sec, 72 °C 15 sec. For vmp3-coll, the first primer pair was vmp3-F5 (5’-GCTTCAAATAAGAATAGCATCAG-3’) / vmp3-R4 (5’-GTTGCTGTATCTGGTGAAGT-3’) followed by vmp3-F4 (5’-CAACTAATT-TTGGACCTAACGG-3’) / vmp3-R3 (5’-GTTTGTAGCTGGTTGATCTGG-3’) and and PCR conditions were 95°C 2 min, 35 cycles of 95°C 30 sec, 55°C 30 sec, 72 °C 1 min. PCR products were analysed on 1.5 % and 0,7 % agarose gel electrophoresis for coll-like and vmp3-Coll, respectively.RESULTS AND DISCUSSIONAll selected CaPsol-associated Bois noir isolates from Croatia showed nested-PCR amplifications with coll-like primer pairs, with three different electrophoretic mobility observed on gel electrophoresis among the eight Croatian samples tested . All selected CaPsol-associated Bois noir isolates from Lebanon showed nested-PCR amplifications with vmp3-coll primer pairs, with at least five different electrophoretic mobility observed on gel electrophoresis among the eighteen Lebanese samples tested. For both genes, the sequencing of the obtained amplicons will indicate the actual number of VNTR repeats. The amplification and sequencing of vmp3-coll VNTR among a set of sixteen CaPsol isolates from eleven Euro-mediterranean countries of the SEE-ERANET/ STOLBUR-EUROMED network (Foissac et al., 2013), indicated that the number of vmp3-coll VNTR was highly variable among CaPsol isolates and ranging from 28 to 64. Only three CaPsol isolates gave no Vmp3-coll VNTR amplifications indicating either the absence of Coll-like and vmp3-coll genes in some of the CaPsol strains tested or sequence variability at the site of primers.ACKNOWLEDGEMENTSThis study was supported by the Plan National Dépérissement du Vignoble grant RENOV and the French-Lebanese bilateral project CEDRE 13E-L2. REFERENCESCimerman, A., Arnaud, G., & Foissac, X. (2006). Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity. Applied and Environmental Microbiology, 72(5), 3274-3283.Cimerman, A., Pacifico, D., Salar, P., Marzachì, C., & Foissac, X. (2009). Striking diversity of vmp1, a variable gene encoding a putative membrane protein of the Stolbur Phytoplasma. Applied and Environmental Microbiology, 75(9), 2951-2957Foissac, X., Carle, P., Fabre, A., Salar, P., Danet, J. L., & STOLBUR-EUROMED-consortium. 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