Using PCR-RFLP for sexing of the endangered Galápagos petrel (Pterodroma phaeopygia)

Abstract

ABSTRACT. Pterodroma phaeopygia is a critically endangered avian species of the Galápagos Islands. This bird is sexually monomorphic, making it difficult to identify the sex. This information, however, is relevant to studies of behavior, ecology, and management of wild or captive populations. Here, we aimed to implement a molecular approach for determining sex in this petrel. DNA was extracted from the blood and the feathers of 24 adult P. phaeopygia, with samples from a female and a male Gallus gallus for comparison. We amplified the cromo-helicase DNA binding protein 1 (CHD-1) gene by PCR, using primers P2 and P8. Allele CHD-1W is unique to females and CHD-1Z occurs in both sexes. We then digested these PCR products using the restriction enzyme HaeIII. The PCR amplified a 400-bp product for both alleles. The digestion of the G. gallus, amplicons split the CHD-1Z allele into two fragments (of 320 and 80 bp), while CHD-1W 4761 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (4): 4760-4767 (2013) Sexing the Galapagos petrel remained intact. Thus, the male exhibited two bands (digested CHD-1Z) and the female three bands (undigested CHD-1W and digested CHD-1Z). Applying this RFLP method on DNA derived from blood, 9 of the 24 petrels were found to be male, while 15 were females. The same results were obtained using feathers as the source of DNA. To our knowledge, this is the first report of molecular method for sexing this species. The potential of sexing this petrel from feathers is remarkable as it minimizes blood sampling induced stress. This method could be used to reinforce the conservation efforts for this bird, to investigate population sex ratios and to develop new conservation strategies.

Derived Polymorphic Amplified Cleaved Sequence (dPACS): A Novel PCR-RFLP Procedure for Detecting Known Single Nucleotide and Deletion–Insertion Polymorphisms

“…This is attested by numerous recent studies that have used PCR-RFLP methodologies both in research and as a routine molecular diagnostic tool. For instance, CAPS assays were successfully developed as potential biomarkers for oral cancer [47], for contributing to a paradigm shift and clearly establishing male heterogamy in two henophidian snakes [48], for molecular sexing in the endangered Galapagos petrel [49] and for ascertaining the identity of the amoebiasis-causing Entamoeba histolytica [50]. The same approach was employed for the rapid diagnosis of neonatal sepsis in India [51] and to determine the magnitude of drug resistance in Mycobacterium tuberculosis in Central Punjab, Pakistan [52].…”

<htm>Improvement of the Molecular Sexing of Parrots in the State of Bahia </htm>

“…The molecular sexing methodology used in the present study has potential applicability in reproductive management and reintroduction programs of birds in the natural habitat. Patino et al (2013) applied the molecular sexing in a management program, where he carried out the translocation of birds of the species Pterodroma magentae (order Procellariiformes) to a predator free area. MARIJA ET AL.…”

Long‐term stability of sex chromosome gene content allows accurate qPCR‐based molecular sexing across birds

“…However, the CHD1 gene is pseudoautosomal in ratites and hence cannot be used as a marker of sex identification in this group (Ellegren, 1996). Furthermore, the CHD1 gene has been used as a marker for the development of alternative PCR‐based molecular sexing methods based on allele‐specific PCR (He et al, 2005; Ito et al, 2003; Lee et al, 2008), restriction fragment length polymorphism (RFLP) (Ellegren, 1996; Bermudez‐Humaran et al, 2002; Patiño et al, 2013; Boano et al, 2020), single‐strand conformation polymorphism (SSCP) (Cortes et al, 1999; Ramos et al, 2009), capillary electrophoresis (Çakmak et al, 2017; Lee et al, 2010), loop‐mediated isothermal amplification (LAMP) (Centeno‐Cuadros et al, 2018; Koch et al, 2019) and real‐time PCR using either TaqMan probes or high‐resolution melting analysis (Brubaker et al, 2011; Chang, Cheng, et al, 2008; Chang, Gu, et al, 2008; Chen et al, 2012; Chou et al, 2010; Faux et al, 2014; Huang et al, 2011; Morinha et al, 2011, 2013, 2019; Rosenthal et al, 2010).…”