Direct PCR assays for DNA barcoding and sexing of plucked feathers

Abstract

The current practice of avian DNA barcoding and molecular sexing involves the use of purified DNA for PCR amplification. The effective use of lysate of minimally invasive plucked feather samples is however questionable for these applications. The present study has aimed at developing a protocol for methodological simplification of avian genetic analysis. The feather lysate was prepared by heating calamus in a dilution reagent at 98 °C for 2–15 min. A total of nine dilution reagents were tested for preparing feather lysates. PBS appeared to be the most efficient dilution reagent for preparing lysate as evidenced by PCR amplification of specific loci from mitochondrial and nuclear genomes. The PCR amplicons of 16S rRNA gene segment from 60 different avian species were subjected to Sanger sequencing. All of these samples were successfully analyzed as a result of one single PCR amplification and sequencing attempt. The PCR amplicons of CHD1 gene segments from Fischer's lovebird were denatured and effectively used for sexual differentiation with high resolution melting (HRM). In sum, direct PCR may be a suitable choice to perform avian genetic analysis using plucked feathers lysed in PBS.