Evaluation of microsatellite markers for the differentiation of specimens and populations of Australian forensically significant blowfly species

DNA-based identification methods have been developed for the accurate identification of forensically relevant arthropods collected from human remains, which is a key factor in the estimation of the postmortem interval. Several DNA regions such as the Cytochrome Oxidase (CO) genes have been studied to evaluate whether the variation observed within these regions can differentiate between forensically important fly species and populations. Although the CO genes have been extensively used in species identification, and a large number of forensically significant fly species can be successfully differentiated using these genes, the ability to differentiate closely related species using these genes is limited. Therefore, there is a need for the identification of genetic makers with sufficient discrimination power at the intra-species level. Microsatellites are repeat motifs of 2-6 bp in the DNA sequence. These repeat units are highly variable among individuals, and thus, are suitable for species and population identification purposes. Microsatellite markers have been developed for forensically significant calliphorids such as Chrysomya albiceps and Cochliomyia hominivorax in Brazil, and Lucilia sericata and Lucilia illustris in Sweden. These microsatellites have been used to identify forensically significant blowflies at the population-level. The use of microsatellites to identify forensically significant blowflies in Australia is scarce. The main aim of this research was to test the suitability of nine previously described microsatellite markers (Torres and Azeredo-Espin, 2008), to differentiate between forensically significant fly species and populations in Australia. Specimens of the species Chrysomya rufifacies, Chrysomya megacephala, Chrysomya varipes, Calliphora dubia, Calliphora albifrontalis, and Lucilia sericata were collected from different sites in Western Australia using liver-baited traps. Additional specimens of these species and Lucilia cuprina and Calliphora vicinia specimens were provided by the QLD Police and the Agriculture Department of NSW. DNA from the thoracic flight muscle of the blowfly specimens was extracted and the quality of the DNA extracts assessed by PCR amplification with an established PCR system. The species of each DNA extract was confirmed by sequence comparison with sequences from the GenBank database. PCR amplification for the microsatellite primer pairs was optimized by varying the annealing temperature and the MgCl2 concentration of each PCR primer pair. The DNA extracts were then amplified with the microsatellite primers using optimized conditions and the PCR products were evaluated using low-resolution gel electrophoresis and medium-resolution MultiNA analysis. No amplification products were obtained with two of the primer pairs. With three of the primers, no large size differences were observed between the species and the populations tested. With the remaining four primers, size differences were observed between the species studied. Further analysis within each population will be necessary to determine if these primer pairs allow species or population differentiation. It is recommended that new microsatellites are isolated from a genomic library obtained from each of the forensically important blowfly species.