Non-invasive PCR-based sex identification using DNA extracted from feces of wild Japanese Rock Ptarmigan Lagopus muta japonica

Abstract

The aim of this study was to establish a non-invasive sex identification method for wild Japanese Rock Ptarmigan Lagopus muta japonica using excreta samples. The utility of preexisting primers was evaluated using blood derived from male and female Svalbard Rock Ptarmigan, L. m. hyperboreus. 2550F/2718R were easily able to distinguish DNA fragments by agarose gel electrophoresis. Then, based on this sequence data, we established a new primer set named Lm-F/Lm-R targeting shorter regions. PCR testing was performed on 132 fecal DNA samples from wild Japanese Rock Ptarmigan, using 2550F/2718R or Lm-F/Lm-R to examine the amplified fragment patterns. 2550F/2718R determined the gender of 68 samples, whereas Lm-F/Lm-R determined 89. The concordance rate (with sexing from the bird appearance like plumage and comb at the time of the collection of these samples) was 88.2% for 2550F/2718R and 85.4% for Lm-F/Lm-R. The amplification rate of the Z-chromosome-derived band was 43.6% for 2550F/2718R and 64.4% for Lm-F/Lm-R. Therefore, the PCR amplification rate was improved by using Lm-F/Lm-R. To be shorter the target amplification region, we were able to improve sex identification.