Development of microsatellite DNA analysis using chicken derived primers for studying gene variation in green peafowl

Abstract

Green peafowl (Pavo muticus imperator) was classified as an endangered species reflected from small population size in Thailand. The great variability in the number of repeats of microsatellite renders these markers suitable in genetic population study. Thus, microsatellite was used to investigate the genetic variation in critical for effective long-term management of natural populations. In this study, DNA templates from green peafowl for microsatellite amplification were extracted from collected bloodstains from wildlife and breeding stations and collected feathers from natural sources and the suitable DNA extraction method from bloodstain and feather specimens were QIAamp® kit and Chelex® method, respectively. For detecting degraded DNA samples, the 3.5-year-old bloodstain samples stored in a desiccator could give amplified PCR products, but the 10-year-old bloodstain samples could not give amplified PCR products. The cross-species amplification in green peafowl was developed by using 23 chicken primers. In optimal PCR condition, 14 primers (60.86%) could be successfully amplify PCR product, only 2 primers (ADL23 and HUJ2) (8.70%) gave allelic polymorphism and the microsatellite motif of the female green peafowl in Phattalung wildlife research and breeding station at the ADL23 locus found to be (CA)₄TA(CA)₂ and at the same locus, (A)n motif found in male and female green peafowl in Khao Soi Dao wildlife research and breeding station. At the LEI80 locus, (CA)₇ motif was found in male green peafowl in Khao Soi Dao wildlife research and breeding station. The (CA)₅GA motif was found at the HUJ2 locus from male green peafowl in Khao Soi Dao wildlife research and breeding station. Touchdown PCR could reduce non-specific bands in the HUJ2 locus and the high allelic polymorphism was found in green peafowl from 7 sites (5 from natural sources and 2 from breeding stations) at the HUJ2 locus. In the case of RAPD, only 2 primers (the primer 1 and 13) from 60 screened primers showed genetic polymorphism. However, the primer 1 gave low allelic polymorphism in green peafowl from 7 sites.