The development of a molecular assay to distinguish droppings of black grouse Tetrao tetrix from those of capercaillie Tetrao urogallus and red grouse Lagopus lagopus scoticus

Counts of faecal droppings are a useful approach for the non-invasive monitoring of species presence and abundance. However, for game birds, it is often difficult to determine unequivocally the species origin of the droppings. Here, we compare the utility of two molecular approaches which exploit variation in the cytochrome b region of mitochondrial DNA to distinguish the droppings of black grouse Tetrao tetrix from those of red grouse Lagopus lagopus scoticus and capercaillie Tetrao urogallus. A sensitive detection method is required as DNA extracts from droppings tend to yield poor quality DNA in low copy number. The first approach adopted a real-time PCR method in which primers were used to amplify a small fragment in the mitochondrial cytochrome b region, and fluorogenic probes complementary to species-specific SNPs were designed. The Success of this test was compared with that of conventional, end point PCR followed by DNA sequencing of a 346 bp mitochondrial cytochrome b region containing the shorter sequence used in the real-time PCR test. In samples which produced results with both the real-time and sequencing approaches, the results were always in agreement. However, the sequencing approach, when used in conjunction with hot-start Taq PCR, proved superior as it worked in a greater number of samples than the real-time method. The length of clear sequence generated allowed secure identification based on several species-specific SNPs. The development of a molecular approach based on a commercially available DNA extraction kit followed by off-site sequencing now offers a secure method of identifying the species origin of field-collected grouse droppings and requires only basic knowledge Of Molecular techniques and inexpensive molecular equipment.