Distinguishing between wild-caught and captive-bred Common Pheasant using methylation rate of skeletal muscle DNA

Abstract

Illegal hunting and trafficking of wildlife and their derivatives extort unprecedented population decline of relatively many species pushing them towards extinction. Notwithstanding contemporary counteracting interventions at international, regional, national and local levels, wildlife farming is advocated as an alternative approach to minimize pressure on wild populations. For wildlife farming to be an effective conservation tool, the integration of wildlife forensics is inevitable to allow distinction between captive-bred and wild-caught species. To this end, we analyzed methylation rates of skeletal muscle samples (pectoralis major, triceps brachii, gastrocnemius, biceps femoris, and neck muscles) from 60 captive-bred and 30 wild-caught Common Pheasant. A total of 13,507 differentially methylated regions were identified between five wild-caught and five captive-bred individuals through whole-genome methylation sequencing (WGBS). Based on the selected five methylation sites, LOC116231076, LOC116242223, ATAD2B, EGFL6, and HS2ST, quantitative detection technique was developed using methylation-sensitive high-resolution melting curve (MS-HRM) to measure methylation rates. The results showed significant differences in methylation rates at all differential sites between wild-caught and captive-bred individuals (|t| = 0.67–33.10, P = 0.000–0.042). The discrimination accuracy rate of each locus was highest in the gastrocnemius muscle and lowest in the neck muscle. The discrimination accuracy rate on LOC116231076, LOC116242223, ATAD2B, EGFL6, and HS2ST methylation sites for gastrocnemius muscle was 64.98%, 100.00%, 68.54%, 63.79%, and 63.70%, respectively; and for neck muscle it was 67.42%, 68.06%, 83.61%, 65.04%, and 68.85%, respectively. The united discrimination accuracy rate of the five loci were 100.00% for gastrocnemius muscle, 99.78% for biceps femoris muscle, 97.52% for pectoralis major muscle, 93.96% for triceps brachii muscle, and 91.63% for neck muscle, respectively. The panel also revealed excellent repeatability, reproducibility, sensitivity and universality to mammals and avian species. This study establishes an effective, accurate and low-cost identification technology for the identification of wild and farmed Common Pheasant, and also provides a reference for the development of identification methods for other species.