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∼79.5 and 77.5 °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.
Huang
Hurng-Wern
HW
Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Su
Ying-Fang
YF
Yao
Cheng-Te
CT
Hung
Yu-Chen
YC
Chen
Chao-Chieh
CC
Cheng
Chien-Chung
CC
Li
Steven Shoei-Lung
SS
Chang
Hsueh-Wei
HW
D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D010856:Columbidae / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012732:Sex Determination Analysis / Q000379:methods*
D030161:Avian Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins