Within the group of the closest relatives of the genus Wolbachia, the sequence of E. ruminantium revealed the highest content of tandem repeats
for bacteria reported so far (Table 4), with size polymorphism in tandem repeats within the isolate that was used for genome sequencing the genome . Our in silico analysis predicted the presence Temsirolimus price of variable tandem repeat mTOR inhibitor review markers in supergroup A strains and could hence readily be developed and tested on Wolbachia isolates from other supergroups. Highly polymorphic markers will be useful in population dynamic and population genetic studies similar to the ones undertaken in wMel-like strains [30, 38, 39]. We have not analysed the unfinished genome data sets of Wolbachia (e.g. ). A large proportion of tandem repeats are located in intergenic regions that tend to be assembled Histone Methyltransferase inhibitor in genome sequencing projects last, yet their conserved flanking regions are required for the isolation of VNTR markers from total genomic extracts. A polymorphic VNTR locus has recently been reported for a supergroup B strain after applying a similar approach to wPip isolated from different C. pipiens populations . Interestingly, our TRF analysis only detected five ANK repeat regions (WD0294, WD0385, WD0514, WD0550 and WD0766) of the 23 annotated
genes encoding ANK repeat domains. Coincidentally, this group of genes includes the most variable genes encoding ANK repeat domains, suggesting that repeat extension/contraction is a strong diversifying mechanism in these genes. Most of the primers designed for wMel ANK genes
amplified expected PCR amplicons from supergroup A Wolbachia, but not from the majority of supergroup B, probably due to sequence divergence . ANK domain genes are known to be present in other Wolbachia groups. In the B group mosquito strain wPip that infects mosquitoes there are 60 genes encoding ANK repeats, some of them also variable Thalidomide [53, 71, 72], whereas the fully sequenced D group wBm strain that infects the nematode Brugia malayi contains 5 ANK genes and 7 related pseudogenes . Although wMel ANK genes were used as a reference in our study, another A group Wolbachia strain, wRi, contains 35 ANK genes, some of them very distinct from the wMel genes, probably as a result of duplications and recombination events . Partial sequences of other A group strains have also revealed high numbers of ANK genes . Thus, it seems clear that ANK genes are a signature feature in Wolbachia that can be potentially utilised to fingerprint closely related strains in A and other groups. Conclusion The identification of amplicon size polymorphic markers of Wolbachia provides a valuable addition to existing typing systems such as MLST, for the following three reasons: (1) The MLVA markers presented here display higher rates of evolution than the MLST loci, which are conserved protein encoding genes.