Initially, work focused on 16S rDNA[16], the genes encoding the cell division protein, ftsZ [11] and the Wolbachia surface protein, wsp [12]. Subsequent to the demonstration of widespread intra- and intergenic recombination betweens strains [17–19], two multi-locus sequence typing (MLST) systems were developed using different sets of a total of 14 Wolbachia genes [20, 21]. The MLST approach uses partial nucleotide sequences of several ubiquitous loci with moderate rates of evolution to generate an allelic profile for tested strains. These profiles can be used to type novel isolates, while the relationships between strains may be inferred on the basis of either the allelic profiles themselves

or the nucleotide sequences underlying them. MLST data have been used for both strain typing and evolutionary NVP-BSK805 purchase Erismodegib order analyses of horizontal transfer events between host species of Wolbachia (e.g. [22, 23]). Since most MLST primer sets cover housekeeping genes that are under purifying selection, these markers often cannot differentiate between closely related strains. Such difficulties have been revealed in the comparisons between wMel, wMelCS and wMelPop [20] or wMel and wAu within the ST-13 complex which appear indistinguishable in MLST loci [21, 24].

These strains induce different phenotypes in their hosts, i.e. wMel induces CI in Drosophila, but wAu does not [25] and wMelPop induces lifespan reduction in its hosts but not wMel [26–28]. The divergence between MLST

typing and actual genomic diversity within ST-13 was also raised when these closely related strains were compared for presence or absence of Wolbachia prophage WO-A and WO-B [24] and other genomic differences such as a large chromosomal inversion and differential IS5 insertion sites between wMel, wMelPop and wMelCS [29, 30]. Furthermore, MLST can be time consuming and expensive for large population genetic studies as it requires sequencing of all MLST loci for many individuals. Recently other typing systems have been developed for bacteria that build on markers that contain during Variable Number Tandem Repeats (VNTR). VNTRs consist of units of DNA (periods) that are tandemly RG7112 repeated and vary in copy number between different isolates. These loci can be used for a PCR-based typing system and are increasingly being utilised in bacterial strain typing such as Multi Locus VNTR Analysis (MLVA) (e.g. [31–35]). MLVA offers a number of advantages, including highly polymorphic markers that allow fine-scale typing of very closely related isolates, rapid, high-throughput screening that is not dependent on sequencing, and potentially the fingerprinting of multiply infected hosts. The modular structure and evolution of these sites through tandem expansion and contraction also allows cladistic and phylogenetic inference.