Screening of the Gamµ-7 Microsatellite Locus to Determine the Sex of Captive Whooping...

Introduction

The whooping crane is North America’s tallest bird species. These birds migrate annually from their breeding grounds in Wood Buffalo National Park in Northern Alberta and the Northwest Territories, Canada, to their wintering range in and around Aransas National Wildlife Refuge north of Corpus Christi, Texas. Whooping Cranes suffered a severe population decline to a low of 14 adults in 1942 ⁽¹⁾ . Subsequent repopulation in Canada has been slow since whooping cranes generally fledge, or raise until able to fly, one chick from the 2–3 fertile eggs laid annually ⁽²⁾ . The whooping crane captive breeding program, initiated in 1967, has relied upon a few captured wild birds and a relatively large number of "excess" eggs collected from cranes at Wood Buffalo National Park ⁽²⁾ .

Figure 1. Whooping crane

Whooping cranes form pair-bonds prior to mating. In captivity, it is important to identify the sex of birds in this pair-bonding species, where synchronous behavioral responses are the cues to mating. Pair-bonds between captive birds can take from months to years to establish, with some pairs never bonding. Because breeding efforts are maximized only after pair-bonds are developed ⁽³⁾ , it is essential to correctly identify sexes of subadult birds to assure the pairings of potential mates are accomplished as early as possible.

Sexing of whooping cranes is problematic because the birds are not sexually dimorphic, and laparoscopy, or "surgical sexing," is stressful for the birds and difficult to assess accurately ⁽³⁾ . Therefore, other less invasive sexing procedures are required. In avian species, females are the heterogametic sex (i.e., the sex that produces gametes containing unlike sex chromosomes, ZW), and are genetically identifiable by the presence of the W chromosome ⁽⁴⁾ . Male birds are homozygous (i.e., carry only one type of sex chromosome, ZZ). Genetic analysis by PCR is quickly becoming the preferred tool for sex determination, where DNA extracted from blood or feathers ⁽⁵⁾ can be used ^{(3) (6) (7)} . Sex determination by DNA fingerprinting or karyotyping ^{(3) (7)} is not attractive because of the associated costs and lengthy analysis time. DNA fingerprinting also demands semi-stringent tissue requirements. Thus, we were interested in developing an approach that was less expensive, quicker, and that could make use of less stringent tissue requirements for sexing whooping cranes ⁽⁶⁾ . We surveyed whooping cranes for allelic variation at more than 20 anonymous microsatellite loci. (Microsatellites or short tandem repeats are sequences found in the genome consisting of 2 to 7 base repeats.) We identified one locus, Gamµ-7, that was sex-linked. Because efforts to sex cranes with the oligonucleotide primers of Griffiths et al. ⁽⁶⁾ resulted in abnormal patterns (unpublished data), we chose to pursue the Gamµ-7 locus for sex identification. In this paper, we describe the use of the Gamµ-7 locus to predict the sex of captive whooping cranes.

Methods

Gamµ-7 genotypes for 91 Whooping Crane DNA samples, obtained from a previous study ⁽⁸⁾ , were tested using the following protocol:

1. PCR amplifications consisted of 1X Taq DNA Polymerase Reaction Buffer (Promega; 50mM KCl, 10mM Tris-HCl [pH 9.0 at 25°C] and 0.1% Triton^® X-100), 1.5mM  MgCl₂, 150µM of each dNTP, 0.6µM of sense and antisense oligonucleotide primers, 1.0 unit Taq DNA Polymerase and approximately 40ng of template DNA in a reaction total of 25µl. The amplification profile consisted of an initial denaturation at 94°C for 3 minutes followed by 30 cycles of denaturation (94°C for 1 minute), annealing (55°C for 1 minute) and extension (72°C for 1 minute). PCR reagents were from Promega.
2. Initially, the amplified microsatellite fragments were separated on a Bio-Rad^® Mini-PROTEAN^® II acrylamide gel apparatus, using 15% acrylamide (19:1 acrylamide to bis- acrylamide), with pBR322/MspI as a fragment size standard.
3. Gels were stained in a solution of ethidium bromide (0.5µg/ml) for 2 minutes, de-stained in distilled water for 10 minutes and then visualized on a UV transilluminator.
4. Accurate fragment size estimation was accomplished by running fluorescently labeled PCR products on an ABI PRISM^® 373 DNA sequencer (Perkin-Elmer) and analyzed using GENESCAN^® software (Perkin-Elmer).
5. Each Gamµ-7 genotype was then compared to a published database containing information on whooping crane sexes.

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Results

Screening of the Gamµ-7 locus by PCR amplification using a sense and antisense oligonucleotide primer pair revealed two distinct banding patterns (Figure 2). The first pattern consisted of three bands, 137, 144 and 150bp, whereas the second pattern consisted of only two bands of 144 and 150bp. The two genotypic profiles were designated "137+" or "137-" in reference to the presence or absence of the 137bp band.

Figure 2. Whooping Cranes exhibit either a "137+" or a "137-" genotype when screened for the Gamµ-7 microsatellite locus.

Genotype electropherograms were produced following PCR amplification of Gamµ-7 PCR products, labeling with FAM fluorophore and application to an ABI PRISM® 373 DNA sequencer (Perkin-Elmer), followed by screening for fragment length with GENESCAN® and GENOTYPER® software (Perkin-Elmer).

Forty of the 41 females exhibited the "137+" profile, whereas 49 of 50 males exhibited the "137-" profile. The DNA of only one individual (#1096) classified as a female failed to produce the 137bp band; likewise, the DNA of only one individual (#1185) classified as a male produced the 137bp band. Re-screening of these two individuals changed #1096's profile from "137-" to "137+", but the second bird (#1185) remained "137+". Laparoscopy was performed subsequently on this bird (#1185) and revealed it to be female. Animals were sexed previously by DNA karyotyping or Z-chromosome-linked markers by restriction fragment length polymorphism.

Discussion

The presence of the 137bp band within known females and its corresponding absence from males suggests that the 137bp band is amplified from the W chromosome. The 144bp fragment is consistent with a duplication on the Z or any autosomal chromosome. The 150bp fragment may be another duplication or a conformational band of the 144bp fragment. It is currently not known which of these fragments corresponds to the original anonymous microsatellite locus identified by Glenn ⁽⁸⁾ . Isolation and sequencing of these fragments will be necessary to determine the relationships of these amplicons.

Whooping crane #1185, which did not fit the reported sex, was listed as a male in the captive breeding population. Anecdotal behavioral and morphometric data from the Calgary Zoo indicated that this bird was male, even though it had never pair-bonded or copulated with its female cagemate. With this bird's DNA profile still representing the (137+) female profile, a decision was made to resex this bird. Laparoscopy confirmed the DNA results, revealing this bird to be female.

Upon re-investigation of the anomalous sex determination, our data demonstrates 100% accuracy of sexing with this microsatellite locus. Using this method, misidentifying a male as female (false positive) is not likely to occur, whereas misidentifying a female as male (false negative) can occur if the amplification of the 137bp fragment is not adequate. We therefore suggest re-amplification of all individuals initially identified as male to prevent potential false-negative results.

We have shown that this microsatellite locus can be used as a quick, accurate and economical sexing tool for whooping cranes. As this study has shown, the conservation of whooping cranes, and by extension other endangered or threatened species, can be enhanced by correcting breeding situations where same-sex birds are mistakenly paired for breeding. The time, effort and money used to promote mating between these two birds can now be redirected to other conservation efforts. Additionally, the genetic diversity of the captive population is increased by allowing these birds a chance to produce offspring. Thus, applying such molecular techniques as demonstrated here should aid in the preservation of this and other unique species.

Acknowledgments

We thank Promega Corporation for the donation of the reagents, which made our study possible. We also thank the Smithsonian Institution's Laboratory of Molecular Systematics for their support during the development of the primers and purification of DNA samples used in this study. Additional funding was provided by U.S. Fish and Wildlife Service, National Fish and Wildlife Foundation, Henry Doorly Zoo, the Audubon Center for Reproduction of Endangered Species, NSF grant DEB-9321604 (to Wolfgang Stephan and Travis Glenn for microsatellite primer development) and Contract DE-AC09-76SROO-819 between the U.S. Department of Energy and University of Georgia's Savannah River Ecology Laboratory.