Figure 6 Diagnostic Pifithrin-�� size polymorphism of the WD0766 gene. Isolates include Wolbachia of D. melanogaster (wMel, wMelCS), D. willistoni (wWil), D. prosaltans (wPro), D. septentriosaltans (wSpt) and D. simulans transinfected with Wolbachia from R.

cerasi (wCer2). A number of inferences about the evolution of the ANK repeats in these genes can be drawn from the tree in Figure 5 and the mapping of the phylogenetic data onto the modular structure of the genes. First, it is likely that the ancestral copy of this gene at the base of supergroup A already contained most of the repeats seen today, probably in a very similar linear order. Most of the clusters in the tree contain repeats from 7 or more of the orthologs, and the order of these orthologous repeats along the genes is highly similar. There is only one clear example of repeat shuffling: the eighth and ninth repeats in the wPro/wSan/wAu groups occur in the reverse order in wCer1 (as repeat periods 10 and 9), while wHa may check details represent an intermediate stage,

with the repeats orthologous to wPro 8 and 9 followed by a second copy of a repeat orthologous to wPro 8. Secondly, at least some variation in repeat number is due to lineage-specific tandem duplication of a single repeat (e.g. repeats 7 and 8 in wCer1) or of multiple repeats (repeats 3-4 and 5-6 in wMel). Extension of MLVA markers to other Wolbachia supergroups In comparison to the MLST markers, the highly polymorphic markers used here have a major trade-off in the loss of universal applicability for all Wolbachia strains. Here we have focused on Wolbachia supergroup A and tested the primers of these markers in other supergroups but primers did not amplify the loci or the loci were not informative. The presence of VNTR loci was restricted to subsets of supergroup A while genes containing

ANK domain repeats were found in all supergroup A strains. In silico analysis of three other completed genomes, wRi, wPip and wBm of supergroups A, B and D, respectively, revealed though that tandem repeated regions occur throughout these supergroups and may be of relevance for MLVA in other supergroups. As further Sirolimus supplier genome data become available it will be possible to extend this to an even larger group of Wolbachia isolates. A TRF analysis of wMel revealed 93 sites with direct tandem repeats of periods ranging from 10bp to 291bp, with internal match percentages from 68% to 100% (Table 4). The larger wRi genome has a similar number of tandem repeats while wPip has a smaller set of tandem repeats. The tandem repeats of wMel, wRi and wPip have similar characteristics such as comparable period sizes, copy numbers as well as internal match ratios (Table 4). The number of tandem repeats in wBm is reduced by a factor of 10 when compared with the supergroup A and B Wolbachia, and the tandem periods appear to be shorter.