Fig. 1

Phylogenetic analysis and sequence alignments of N. benthamiana ubiquitin E2 genes. A Phylogenetic analysis of 40 N. benthamiana ubiquitin E2 genes. The DNA sequences of the open reading frames (ORFs) of 40 N. benthamiana E2 genes were used for generating the tree. We used the DNA sequences but not protein sequences to generate the phylogenetic tree to identify most potential off-targets when silencing group III E2 genes using VIGS. The unrooted phylogenetic tree was generated by the neighbor-joining method using the MEGA6 program with 1000 bootstrap trials [61, 68]. The phylogenetic tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The Roman numerals designate the different E2 groups that were based on the phylogenetic analysis of protein sequences as shown in Additional file 1: Fig. S1. B Phylogenetic analysis of group III E2 genes. The DNA sequences of the open reading frames (ORFs) of group III E2 genes were used for generating the tree. Same parameters as described in A were employed for the phylogenetic analysis. The letters (a–f) designate the six subgroups of group III. C Sequence alignments of group III E2 genes. The DNA sequences of the open reading frames (ORFs) of group III E2 genes in the FASTA format were entered into MultAlin and aligned using the default parameters [59]. Color black denotes high consensus nucleotide while red denotes low and neutral consensus nucleotide