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Sex Determination of Owls through the Genotyping of Buccal Samples
Laura Traut, Hannah Salk, Dr. Kelsey Metzger
Background and Significance
Results and Discussion
Conclusions and Future Direction
Methods
References
• DNA extraction of avian buccal samples
-Samples were collected with OmniSwabs and stored in TE buffer
-Extraction performed using Qiagen QiaAmp DNA Mini Kit
• Polymerase Chain Reaction performed to amplify CHD1 and XHO1 genes
-XHO1 primer used (Smith, 1997) Forward: 5’ATC TAC CAC TTT TCT CAG GG3’
Reverse: 5’TTC AGA GTG ATA ACG CAT GG3’
-CHD1 primers used (Ellegren, 1999) Forward: 5’GTT ACT GAT TCG TCT ACG AGA3’
Reverse: 5’ATT GAA ATG ATC CAG TGC TTG 3’
-Initial denature: 94°C for 5 minutes
-35 cycles of denature (95°C for 30 sec), anneal (55°C for 30 sec),
extend (72°C for 30 sec)
-Final extension: 72°C for 5 minutes & hold at 4°C until frozen at -20°C
• Gel Electrophoresis
-Ladder used: pBR322/BSTN1 (1857, 1058, 929, 383, & 121 bp)
-Run for 15 minutes
-Results analyzed using ultraviolet light
• The CHD1 gene is W and Z linked, but
the genes are of different sizes. In males
(ZZ), we expected to see one band in gel
electrophoresis at 600-650 bp. In
females (ZW), we expected to see two
bands: one at 400-450 bp and one at
600-650 bp.
• The XHO1 gene is present only on the W
chromosome, so we only expected to
see one band at 168 bp in the female
owls and no bands in the male owl
samples.
• Avian species have ZW/ZZ sex determination systems
• Females possess ZW sex chromosomes while males possess ZZ sex chromosomes
• Quarry Hill Nature Center (QHNC) provided 9 buccal samples for sex determination through genotyping (7 samples with sex known, and 2 who are unknown).
QHNC tracks species of migratory birds and identifies individual birds through leg banding, sex, and size.
• Amplification, through polymerase chain reaction, of CHD1 and XHO1 genes followed by gel electrophoresis will allow for identification of sex for the 2 unknown owl
samples
• Previous uncertainties surrounding effectiveness of using XHO1 gene as sex identifier
• Using the CHD1 gene will allow us to compare the results of the two different gene amplifications
CHD1 Gel Electrophoresis
• Lane 5 shows 2 distinct bands
• Lanes 6, 7, and 8 show smearing
bands. This may indicate the presence
of 2 CHD1 genes of different sizes.
• Results suggest samples in lanes 5,
6, 7, and 8 belong to female avian
individuals. These results also suggest
samples in lanes 2, 3, 4, 9, and 10
belong to male avian individuals.
• Lane 11 shows no banding,
indicating non-contaminated DNA
samples
XHO1 Gel Electrophoresis
• Lanes 6, 7, and 8 show distinct
bands.
• Results suggest samples in lanes
6, 7, and 8 belong to female avian
individuals. These results also
suggest samples in lanes 2, 3, 4, 5,
9, and 10 belong to male avian
individuals. Fridolfsson, A. K., & Ellegren, H. (1999). A simple and universal method for molecular sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121. Smith, C. A., Andrews, J. E., & Sinclair, A. H. (1997). Gonadal sex differentiation in chicken embryos: expression of estogen receptor and aromatase genes. Steroid Biochemistry Molecular Biology, 60(5-6), 295-302.
Avian Species and Sample # Actual Sex Predicted Sex Gene(s) Indicating Sex
Aegolius acadicus #101-Lane 2
*Unknown Male CHD1 and XHO1
Aegolius acadicus #102-Lane 3
*Unknown Male CHD1 and XHO1
Falco sparverius #103-Lane 4
Male Male CHD1 and XHO1
Megascops asio #104-Lane 5
Female Female CHD1
Gallus gallus domesticus #105-Lane 6
Female Female CHD1 and XHO1
Gallus gallus domesticus #106-Lane 7
Female Female CHD1 and XHO1
Gallus gallus domesticus #107-Lane 8
Female Female CHD1 and XHO1
Gallus gallus domesticus #108-Lane 9
Male Male CHD1 and XHO1
Gallus gallus domesticus #109-Lane 10
Male Male CHD1 and XHO1
Results from gel electrophoresis of both gene amplifications identify the 2 unknown
samples as male. XHO1 seemed to adequately identify avian sex.
For future direction, re-amplification of the XHO1 gene for sample #104 would be
beneficial to determine if there was human error, amplification issues, non-optimal
annealing temperature, etc. We would also like to determine why banding in lane 5 of
the CHD1 gene did not appear in the predicted locations.
Predictions