Chien-Chung Cheng

An improved PCR method for gender identification of eagles

Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay.

High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes

The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

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.

Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species

The objective of this study was to test the hypothesis that genders of Accipitridae species, with the same or similar sequences to our previously proposed Spilornis cheela hoya (S. c. hoya) chromo-helicase-DNA binding protein (CHD)-W-specific and CHD-ZW-common TaqMan probes, can be successfully determined. Eight species of Accipitridae with known genders were collected. After PCR, TA cloning, sequencing, and alignment analyses, sequence length differences of Griffiths P2/P8 PCR amplicons between CHD-Z and CHD-W genes ranged from 2 to 19 bp for these Accipitridae species, and they were unsolved in 3% agarose gel. Using our previous proposed S. c. hoya TaqMan probes, the genders of Circaetus gallicus, completely homologous to the sequences for these CHD probes, were successfully identified. With one nucleotide difference to S. c. hoya CHD-W-specific probe, gender identification of Accipiter gularis, Accipiter soloensis, Accipiter trivirgatus, Accipiter virgatus, and Butastur indicus were validated. With two nucleotide differences in the CHD-W-specific probe and one nucleotide difference in the CHD-ZW-common probe, Pernis ptilorhyncus also performed well for gender identification. In conclusion, the S. c. hoyaCHD probes, coupled with the Griffiths P2/P8 primers, were validated to provide accurate and high-throughput gender identification for many Accipitridae species.

High-throughput sex identification by melting curve analysis in blue-breasted quail and chicken

The objective was to develop a high-throughput method of identifying sex in both Coturnix chinensis and Gallus gallus, which would be useful for biomedical research and hatcheries. Because chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primers do not produce polymerase chain reaction (PCR) products with distinguishable sex-specific curves in melting curve analysis (MCA), these primers are unsuitable for high throughput application in either species. Conserved regions were identified by basic local alignment search tool (BLAST) analyses of cloned CHD-Z and CHD-W genes of C. chinensis. Based on sequence alignment, a female-specific CHD-W primer (W-cot-F1) and a female/male (or CHD-W/CHD-Z)-common primer (ZW-cot-F1) were redesigned for use in combination with the Griffiths P2 primer for MCA-based PCR reaction. In C. chinensis and G. gallus, W-cot-F1/P2 and ZW-cot-F1/P2 had amplicon lengths of 315/318 and 114 base pairs and melting temperatures (Tm) of approximately 79.5 °C to 80 °C and approximately 78.5 °C to 79°C, respectively. Thus, MCA distinguished sex based on two distinct Tm peaks in females versus only one Tm peak in males. The MCA-based real-time PCR combined with the proposed primer redesign provided a high-throughput method of identifying sex in C. chinensis and G. gallus.

High-throughput gender identification of penguin species using melting curve analysis.

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 Griffithss 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 Griffithss 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.