title: High-throughput gender identification of penguin species using melting curve analysis.
abstract: Most species of penguins are sexual monomorphic and therefore it is difficult to visually identify their genders for monitoring population stability in terms of sex ratio analysis. In this study, we evaluated the suitability using melting curve analysis (MCA) for high-throughput gender identification of penguins. Preliminary test indicated that the Griffiths's P2/P8 primers were not suitable for MCA analysis. Based on sequence alignment of Chromo-Helicase-DNA binding protein (CHD)-W and CHD-Z genes from four species of penguins (Pygoscelis papua, Aptenodytes patagonicus, Spheniscus magellanicus, and Eudyptes chrysocome), we redesigned forward primers for the CHD-W/CHD-Z-common region (PGU-ZW2) and the CHD-W-specific region (PGU-W2) to be used in combination with the reverse Griffiths's P2 primer. When tested with P. papua samples, PCR using P2/PGU-ZW2 and P2/PGU-W2 primer sets generated two amplicons of 148- and 356-bp, respectively, which were easily resolved in 1.5% agarose gels. MCA analysis indicated the melting temperature (Tm) values for P2/PGU-ZW2 and P2/PGU-W2 amplicons of P. papua samples were 79.75°C-80.5°C and 81.0°C-81.5°C, respectively. Females displayed both ZW-common and W-specific Tm peaks, whereas male was positive only for ZW-common peak. Taken together, our redesigned primers coupled with MCA analysis allows precise high throughput gender identification for P. papua, and potentially for other penguin species such as A. patagonicus, S. magellanicus, and E. chrysocome as well.
authors.affiliation: a Graduate Institute of Natural Products, College of Pharmacy , Kaohsiung Medical University , Kaohsiung , Taiwan.
authors.forename: Chao-Neng
authors.initials: CN
authors.lastname: Tseng
authors.forename: Yung-Ting
authors.initials: YT
authors.lastname: Chang
authors.forename: Hui-Tzu
authors.initials: HT
authors.lastname: Chiu
authors.forename: Yii-Cheng
authors.initials: YC
authors.lastname: Chou
authors.forename: Hurng-Wern
authors.initials: HW
authors.lastname: Huang
authors.forename: Chien-Chung
authors.initials: CC
authors.lastname: Cheng
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Liao
authors.forename: Hsueh-Wei
authors.initials: HW
authors.lastname: Chang
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D005820:Genetic Testing / Q000379:methods; D008297:Male; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012727:Sex Characteristics; D012732:Sex Determination Analysis / Q000379:methods*; D013045:Species Specificity; D046429:Spheniscidae / Q000145:classification* / Q000235:genetics*; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins