An improved PCR method for gender identification of eagles
Introduction
Eagles, hawks, harriers, vultures, and buzzards are the common names for the birds belonging to the nine subfamilies of the family of Accipitridae, order Falconiformes [1]. All of them are at the top of ecological food webs and they are very sensitive to environmental changes such as chemical contaminants, food shortages, sex ratio distortion, and habitat destruction. They represent one of the most important ecological indicators of environmental health.
One feature of these birds is that they do not exhibit any external sexual dimorphism. The males and females show only slight differences in their body size [1]. Therefore, it is difficult to determine the sex ratio in a population, which is a crucial parameter for studies of population biology and conservation. The difficulty of avian gender identification may hinder both evolutionary studies and human-assisted breeding of wildlife, especially for endangered species. Thus, developing a rapid, economical, and reliable method for eagle gender identification is important, not only for species management but also to assess environmental impacts.
Many techniques currently used for gender determination of monomorphic birds have been recently reviewed [2] but most of these are time-consuming, expensive, or may harm the animals to be tested. In light of these caveats, PCR-based sex identification provides a solution to circumvent these problems [3]. Gender assays in birds are generally based on the CHD-W gene, located on the W chromosome and therefore unique to females. Its homolog, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). PCR-based gender identification employs a single set of primers, which amplifies homologous sections of both genes and incorporates introns whose lengths usually vary among species [4], [5], [6]. Although CHD-Z and/or CHD-W can be PCR-amplified [7], [8] using a universal CHD primer pair P2/P8 [6], increasing lines of evidence [4], [7], [9], [10], [11] have shown that the genders of some avian species cannot be determined by this protocol.
In this study, we evaluated the suitability of the currently used P2/P8 primers and improved upon them for the accurate identification of gender in Spilornis cheela hoya. More importantly, our redesigned primers are conserved for at least 10 species of eagles including species from different genera and may make this protocol of broader use.
Section snippets
Sample collection and blood DNA extraction
Twenty-four blood samples (Bd1–24) from banded S. cheela hoya were collected under official permission of the Kenting National Park, Taiwan. Based upon their behavior during incubation, brooding and feeding, Bd1 and 15 were designated as female controls. Blood DNA was extracted by a Qiagen blood kit as previously described [12]. Also, four tissue samples of S. cheela hoya (males: 4655 and 4966; females: 4968 and 5151) with anatomically confirmed genders were provided from the Taiwan Endemic
Gender identification problem for S. cheela hoya using P2/P8 universal primers
In Fig. 1, the electrophoresis of PCR products using the P2/P8 primers on a 4% agarose showed a single band regardless of gender in 10 individuals of S. cheela hoya. After careful examination, we found that there are actually two PCR-amplified bands with different MWs: the lower MW band was found in Bd3–5, 7, 9 and 10, while the higher band was presented in Bd1, 2, 6 and 8. To address the reason underlying the failure of gender identification using the P2/P8-based PCR test in S. cheela hoya,
Discussion
Population sex ratios in monogamous birds are often male-biased [16], [17], [18]. In order to achieve successful breeding programs, it is essential to know the sex ratio of eagle populations. Traditionally, the Griffiths procedure for avian molecular gender identification using the P2/P8 primers [6] is dependent on chromosome-specific intron size differences [5] of the avian CHD genes. However, certain problems are inherent to this assay. For example, some species of birds show limited
Conclusion
We have developed an improved PCR assay for gender identification in eagles that is easily performed using readily available techniques. The technique is likely to be an important tool in the management of populations of these important species.
Acknowledgements
This work was supported in part by the National Science Council in Taiwan under the grants NSC 96-2622-B-037-003-CC3 and NSC95-2622-B-037-002-CC3 to H-W Chang. The authors are also grateful to the help from the Kenting National Park, Taiwan.
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2023, Forensic Science International: Animals and EnvironmentsApplication of Two Molecular Sexing Methods for Indonesian Bird Species: Implication for Captive Breeding Programs in Indonesia
2012, HAYATI Journal of BiosciencesCitation Excerpt :The P8/P2 primers amplified two alternative PCR fragments, but the size difference between two fragments were too short. Because the length in P8/P2-amplified Z- and W-fragments, which are extremely short, making it hard to resolve them on agarose gels (Chang et al. 2008). According to Fridolfsson and Ellegren (1999), the difference in size between Z- and W-spesific fragments amplified with the P8/P2 primers, ranging 10-80 bp.
High-throughput sex identification by melting curve analysis in blue-breasted quail and chicken
2012, TheriogenologyCitation Excerpt :The sex of birds can generally be determined by comparing intron length between the chromo-helicase-DNA binding protein in (CHD)-Z and CHD-W genes. Many polymerase chain reaction (PCR)-based methods of molecular sex identification have been developed [17–24]. The conventional sex identification methods used in quail are 2718R/2550F [17,25] and P2/P8-based PCR-restriction fragment length polymorphism (RFLP) [26].
High-throughput gender identification of three Columbidae species using melting curve analysis
2011, TheriogenologyCitation Excerpt :Therefore, we hypothesized that the MCA can be used for gender identification of some Columbidae species such as Columba livia (C. livia), Columba pulchricollis (C. pulchricollis), and Streptopelia tranquebarica (S. tranquebarica). Although MCA may be of use to determine Tm values for CHD-Z and CHD-W amplicons, the lengths between them differ among species [14,26]. If it is unsolved in MCA, it is necessary to redesign other primers to increase the length difference of intron between amplicons for CHD-Z and CHD-W genes.
Molecular phylogeny of the Pycnonotus sinensis and Pycnonotus taivanus in Taiwan based on sequence variations of nuclear CHD and mitochondrial cytochrome b genes
2010, Biochemical Systematics and EcologyCitation Excerpt :Briefly, DNA samples were added to the PCR reaction mixture (10 μl) containing 1 μl of 10× PCR buffer, 0.3 μl of 50 mM MgCl2, 0.2 μl of 10 mM dNTP each, 0.6 μl DMSO, 0.14 μl of Taq enzyme, 0.12 μl of 350 μg/ml primers mix (1:1), and 7.64 μl of DNA in water. The PCR program is as follows: 94 °C for 4 min, 4 cycles of 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 30 s; 49 cycles of 94 °C for 30 s, 46 °C for 20 s, and 72 °C for 20 s; and a final extension at 72 °C for 5 min (Chang et al., 2008a,b). PCR product was detected by running in 1.5% agarose gel electrophoresis at 100 V for 30 min and visualized with ethidium bromide (EtBr) staining.