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