PCR-Based Method for Sexing Roseate Terns (Sterna dougallii)

Abstract

A simple method for identifying the sex of sexually monomorphic birds would be widely applicable to studies of population-genetic structure, demography, and behavior. In this paper we describe a method for identifying W-chromosome-specific genetic markers and show that these markers can be used to identify sex from very small samples of tissue, such as single mature feathers and, thus, from individuals of any age. We have applied this technique to Roseate Terns (Sterna dougallii) and further shown that the marker correctly identified sex in a closely related species, the Common Tern (Sterna hirundo). This method can be used to develop markers for other groups of birds, or any organism with chromosomal sex determination. The Roseate Tern is a cosmopolitan species of seabird that occurs in widely separated breeding populations in temperate and tropical regions of the Atlantic, Indian, and West Pacific oceans (Cramp 1985). The population in the northwestern Atlantic, which is the subject of our study, is listed as endangered (U.S. Fish and Wildlife Service 1987). Preliminary observations indicate a female-biased sex ratio in this population (Hatch and Nisbet pers. obs.). Information about the sex of many more individuals at various life cycle stages will be important for characterizing sex-specific behavior and investigating this femalebiased population sex ratio. Like many terns and other seabirds, the Roseate Tern is sexually monomorphic in plumage, and the size differences between males and females are small and not well characterized. Sex-specific behaviors (copulation, courtship feeding) in this species are usually performed away from the nest site and only occasionally can be used to identify the sex of individual birds. Alternative methods for sexing include laparoscopy and handling the adults during the period of egg laying when morphological differences are distinct. However, both of these methods pose unacceptable risks of injury or nest abandonment for members of this endangered population. Another method for sexing birds based on analysis of tissue samples is karyotyping from fibroblasts grown from blood-feather pulp (Parker et al. 1991). This method is laborious and has not been used on terns. Molecular techniques for sexing used on several bird species have been based on hybridizing labeled probes to genomic DNA. The probes are either W-chromosome-specific or they identify multigene families with W-specific members (Griffiths and Holland 1990, Quinn et al. 1990, Dvorak et al. 1992, Griffiths 1992, Millar et al. 1992, Graves et al. 1993, Longmire et al. 1993). These molecular methods are slow and laborious, they require relatively large quantities of DNA, and most of them involve use of radioisotopes. Such methods are neither economical nor practical for field studies involving large sample sizes or nestlings. With the advent of random-amplified polymorphic DNA (RAPD; Williams et al. 1990), a new source of easily accessible genetic markers is available. These markers have been applied to genetic studies in plants (Tingey et al. 1992) and, recently, have been used for sexing birds (Griffiths and Tiwari 1993). We describe a method for sexing Roseate Terns based on the polymerase chain reaction (PCR) that offers considerable advantages in technical simplicity and time. The method was developed with blood samples from adult Roseate Terns. However, because only nanogram quantities of DNA are required, the method is much more versatile and works with single mature feathers. Genetic markers specific to the W chromosome are identified by separately pooling samples of male and female DNA, and then screening the two pools for RAPDs (Michelmore et al. 1991). A typical RAPD reaction yields 6 to 10 bands (loci) that are produced by amplification with one arbitrary 10-base primer. Once a W-linked RAPD marker has been identified, it can be converted to a more specific and reliable PCR-based marker (i.e. a sequence-characterized amplified region, SCAR; Paran and Michelmore 1992) by increasing the length of the original primer at each end of the targeted region. The high specificity of a SCAR marker overcomes several of the drawbacks of RAPDs (Kesseli et al. 1992, 1994, Paran and Michelmore 1992). Methods.-Roseate Terns initially were sexed using an RFLP probe (Zoogen Inc., Davis, California). The method was verified using field-sexed Common Terns. The Roseate Terns sexed by this method were used to identify RAPD markers and develop SCAR primers. Finally, these primers were tested on a larger sample of field-sexed Common Terns. Terns were trapped at nests on Bird Island, Marion, Massachusetts (41?40'N, 70?43'W). Roseate Terns were trapped shortly before their eggs hatched. Common Terns were trapped within 24 h after the first egg was