High-throughput gender identification of three Columbidae species using melting curve analysis

The objective was to perform high-throughput gender identification of three Columbidae species (Columba livia, Columba pulchricollis, and Streptopelia tranquebarica). Although the chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primer set resolved the amplicon products of these species in 3% agarose gel electrophoresis, it was unsuitable for molecular gender identification using the melting curve analysis (MCA) curve for high-throughput analysis. After sequencing the CHD-Z and CHD-W genes for these species, we redesigned a female-specific CHD-W primer (dove-W) and a female/male (or CHD-Z/CHD-W)-common primer (dove-ZW) to combine with the Griffiths P2 primer to generate two PCR amplicons with different lengths (P2/dove-W and P2/dove-ZW for 252- and 104-bp, respectively). Melting temperature (Tm) values for P2/dove-W and P2/ dove-ZW amplicons were determined and resolved in MCA at approximately 79.0 similar to 79.5 and 77.5 degrees C, respectively. Accordingly, females contained two Tm peaks, whereas males contained one. In conclusion, melting curve analysis (MCA) using our proposed primer sets was a robust gender identification method for the three Columbidae species tested. (C) 2011 Elsevier Inc. All rights reserved.