Development of microsatellite marker for identifying Japanese squirrelsNoninvasive genetic sampling on the road

The arboreal mammals are potentially highly vulnerable to discontinuities in habitat created by road. There is a scarcity of research and survey methods regarding arboreal mammals in road ecology. In this study, we carried out the selection of microsatellite markers to identify individuals from trace DNA in order to apply to the pre- and post-assessment on road of arboreal mammals. We developed an effective method for collecting trace DNA from food marks samples of wild individuals in the field. We extracted trace DNA from the collected food marks samples and verified the possibility of identifying individuals in the field.

Using 52 DNA samples of Japanese squirrels, we evaluated the polymorphism of total 63 microsatellite markers of squirrel genus. From a set of 37 microsatellite markers, we selected 10 individual discrimination markers for Japanese squirrels i.e. Lis-09, Scv-15, Scv-04, Scv-16, FO-33, FO-35, FO-36, FO-39, FO-4 and FO-48 based on the lowest possibility of identity values (PI-sibs).

DNA was extracted from non-invasive samples such as feces or saliva can be of low quantity, low molecular weight and often contaminated with PCR inhibitors. Taking this into consideration, we used feces samples and food trace samples expected to contain saliva of Japanese squirrels, and carried out this study in three parts: 1) sample storage method; 2) DNA extraction method; and 3) Efficient PCR reaction conditions. Optimal analysis conditions for individual identification method of Japanese squirrels with non-invasive samples were determined.

We collected a total of 45 of feces and food traces samples (apples and sweet potatoes) from Japanese squirrels bred in Inokashira natural culture park and analyzed individual identification using the selected 10 microsatellite markers. Individual identification was successful in 28 out of 45 samples, and the success rate was 62.2%. The success rate by sample type, fecal samples were 89.5% (successful in 19 samples out of 17 samples), food samples (apple) were 12.5% (success in one sample out of 8 samples), and food samples (sweet potato) were 55.6% (18 samples in success in 10 samples). Fecal samples showed the highest success rate.

We analyzed a total of 29 food traces (pine cone) of Japanese squirrels collected at Kashi road (about 1.0km × 1.5km around Fukushima Prefecture’s Minamiaizu-gun Shimogo-machi) for identification using the 10 microsatellite markers. Individual identification was successful in 5 samples out of 29 samples, and the success rate was

Identification success rate was low from food traces (Pine cone) in the field.

The success rate of fecal samples is the highest. But we could sufficiently identified individuals in the field using DNA extracted from saliva.

In order to capture saliva easily, we suggested that we set up bait traps and capture food trace with saliva.

Microsatellite DNA;Japanese squirrel;Noninvasive genetic sampling