## 4 Discussion  
The aim of this study was to investigate the roles that _Vitis vinifera_ cultivars might play, as microbial hosts, in shaping the differentiation of associated microbiomes. The hypothesis in the study was that if there is any influence of the genotype in the bacterial assemblage that overwinter on the vine canes, this should be evident as differences in the bacterial populations detected early in the spring on vines of different genotypes in the same vineyard, same soil and environment, and cultivated with the same agricultural practices. Here, we specifically focused on one-season-old canes' barks and buds, which to our knowledge, no other study compared their associated microbiomes yet. High-throughput sequencing revealed rich microbiomes on these parts from several cultivars, supporting that trunk bark is an important microbial host (microbial terror [7]). Interestingly, cane samples hosted several taxa that are typically identified in many studies across the world. On the other hand, a great portion of our sequencing data revealed unclassified microbial sequences. However, it is not uncommon for sequencing studies to result in high numbers of valid but unclassified reads. It could be due to experimental noise and richer microbiomes with some organisms less well described in the different databases [31]. Also, the differences in the microbiomes of the studied cultivars partly support the idea that vineyards consisting of different grape cultivars appear to harbor more diverse microbiomes. A possible selection pressure imposed on the microbiome by grapevine genotype might be one explanation, as indicated in other studies [4]. Figure 5: The relative abundance of the top 11 bacterial genera among the 37 cultivars. A multiple variable region 16S amplicon approach was applied to target seven hypervariable regions of the 16S rRNA gene in Vitis microbiomes, contrary to other previous studies focusing on a single region [4; 6; 7; 11; 12; 16; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50]. The use of multiple variable regions of the bacterial 16S rRNA gene provides clear advantages compared to traditional single variable region approaches, particularly regarding detection specificity, and can better describe microbial diversity, i.e., higher resolution for lower-rank taxa [27; 51]. A multiple variable region 16S amplicon approach could be incorporated in current workflows of Illumina [27] and Thermo Ion sequencing technologies (Ion 16S Metagenomics Kit, #A26216, Thermo Fisher Scientific Inc., USA) without any subsequent modifications after amplicon amplification and purification. Among the seven hypervariable regions of the 16S rRNA gene analyzed, the V3 region provided the greatest sequencing depth for profiling bacterial diversity, followed by V6-7 and V4. The later region (V4) also provided greater sequencing depth compared to the V5 region in another Vitis study [46]. Therefore, sequencing V3-4 regions, with high specificity primers excluding co-amplification of mitochondrial/chloroplast DNA, could be an effective alternative for future _Vitis_ related studies. Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla. These phyla are considered as phyllosphere-associated generalists and have been detected in large numbers in several plant species [52; 53] and soil samples as well [54; 55]. Although their dominance detected in our study was in accordance with the findings of others [4; 7; 11; 12; 18; 37; 38; 39; 40; 44; 45; 56; 57; 58; 59; 60; 61], however, their niches were entirely neglected. The rest of the phyla that had a low presence were also detected in other studies in different amounts. For instance, the prevalence of cyanobacteria was very low (0.5%) compared to 80% reported by [4], who examined phyllosphere and carosphere, probably due to the different tissues examined.