Establishing paternity in whooping cranes (Grus americana) by DNA analysis

-DNA fingerprinting was used to study paternity and genetic variability within a captive flock of Whooping Cranes (Grus americana). Fingerprint patterns for 42 individuals were obtained by digesting genomic crane DNAs with HaeIII followed by electrophoresis, blotting, and hybridization to the M13 minisatellite probe. Despite finding reduced levels of genetic variation in the Whooping Crane due to a population "bottleneck," these polymorphisms were successfully used to determine paternity in six of seven cases of captive propagation where the maternal-offspring relationship was known, but where the sire was unknown. These determinations of paternity are required for effective genetic management of the crane flock. These results also revealed a number of heterozygous minisatellite loci that will be valuable in future assessments of genetic variability in this endangered species. Received 10 July 1991, accepted 10 February 1992. THE WHOOPING CRANE (Grus americana) is a highly endangered species. Today's 221 surviving individuals are all descendants of a single natural population that nests in Wood Buffalo National Park, Canada, and over-winters at Aransas National Wildlife Refuge near Austwell, Texas. The wild Wood Buffalo population reached a low of 15 birds in 1941 and then slowly increased to 48 in 1967, and to 142 in 1990 (Erickson 1968, Ellis et al. 1991). Besides the 142 cranes making up the Wood BuffaloAransas population, in 1990 there were 66 individuals in captivity and 13 in an experimental transplant-release program in Idaho. Due to its endangered status, the Whooping Crane has been the subject of intense efforts aimed at preservation and recovery of the species. These efforts have included ecological study, habitat management, and reintroduction (U.S. Fish and Wildlife Service 1986, Canadian Wildlife Service 1988). A captive propagation program was initiated at the Patuxent Wildlife Research Center in Laurel, Maryland, in 1965. Relying principally on artificial insemination, this project has produced 50 captive-reared cranes since 1975, and has supplied 73 fertile eggs for transplant release. Whooping Cranes represent a substantial captive investment due to slow sexual maturity and low reproductive capacity (only 60% of eight-year-old captive females have produced eggs, and only five or six eggs are produced per female per year). In order to maximize the production of fertile eggs, adult females often were inseminated with semen from several males. In these cases, the female parent was known, but the sire was not known. The unknown paternities pose a significant impediment to future breedings. In order to avoid the possibility of inbreeding, female offspring could not be mated with any of their possible sires. In addition, it was not possible to distinguish sibling and half-sibling relationships among these offspring. Determining paternal relationships, thus, would benefit those involved in the genetic management of the captive Whooping Crane flock. Although knowledge of paternity has been needed for some time, conventional techniques (such as allozyme analysis) have failed to resolve paternity in most birds, including these cranes. However, the recent development of DNA fingerprinting (or DNA profiling) has made it feasible to perform meaningful paternity testing in avian species. This technique makes use of "minisatellites," which are short tandemly repeated DNA sequences. An interesting feature of minisatellites is that the nu-