Bird sex-typing using genetic markers




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Sheffield Molecular Genetics Facility

Bird sex-typing using genetic markers

by Deborah Dawson (D.A.Dawson @Sheffield.ac.uk)



Technical Details

For the confident assignment of sex in non-ratite birds we recommend that both the Griffiths et al. (1998: P2/P8) and Fridolfsson and Ellegren (1999: 2550F/2718R) sexing primers should initially be tested in order to identify the primers best suited to the study species. 



Primer sequences:

Griffiths et al. (1998: P2/P8)


 P2 = 5'-TCT GCA TCG CTA AAT CCT TT-3'
 P8 = 5'-CTC CCA AGG ATG AGR AAY TG-3'

Fridolfsson and Ellegren (1999: 2550F/2718R)


 2550F = 5'-GTT ACT GAT TCG TCT ACG AGA-3'
 2718R = 5'-ATT GAA ATG ATC CAG TGC TTG-3'

The usual protocol adopted by the Sheffield Molecular Genetics Facility is detailed here.

DNA was extracted from blood following Bruford et al. (1998). Each 10-µl reaction contained about 50 ng of genomic DNA, 1 µM of each primer and 0.25 units Taq DNA polymerase (ThermoprimePlus, Advanced Biotechnologies) in the manufacturer’s buffer (final concentrations 20 mM (NH4)2SO4, 75 mM Tris-HCl pH 9.0, 0.01% (w/v) Tween), including 2.0 mM MgCl2 and 0.2 mM of each dNTP. PCR amplification was performed in a Hybaid Touchdown thermal cycler. 

If the concentration of high quality DNA is low (eg if DNA has been extracted from feathers) the following program should be used (the annealing temparature has been reduced, time at eact temperature and the number of cycles have been increased). In many cases this program is used as the first choice:



The reaction profile used with the Griffiths et al. (1998) P2/P8 primers was:-

94°C 2 minutes 


then: 
94°C 45 seconds
48°C 45 seconds
72°C 45 seconds
for 40 cycles,

followed by:


72°C for 5 minutes

The reaction profile used with the Fridolfsson and Ellegren (1999) 2550F/2718R primers was:-

94°C 2 minutes


then: 
94°C 30 seconds
60°C 30 seconds (reducing by 1°C each cycle), 
72°C 30 seconds
for 1 cycle, 

94°C 30 seconds


59°C 30 seconds (reducing by 1°C each cycle), 
72°C 30 seconds
for 1 cycle, 

etc, reducing annealing temperature by 1°C each cycle until..

94°C 30 seconds 
51°C 30 seconds (reducing by 1°C each cycle), 
72°C 30 seconds
for 1 cycle, 

followed by:


94°C 30 seconds 
50°C 30 seconds 
72°C 30 seconds
for 30 cycles. 

followed by:


72°C for 5 minutes

The following profile used with the Griffiths et al. (1998) P2/P8 primers has been used in the past and works well for many species: 94°C 2 min then 94°C 15 s, 50°C 20 s, 72°C 25 s for 40 cycles followed by 72°C for 1 minute.

The PCR products were visualised either on 3% agarose gels stained with ethidium bromide, or on 5-6% polyacrylamide gels stained with silver (Promega) or analysed on an ABI 377 DNA sequencer.

We would also like to draw attention to the potential difficulties in CHD-based molecular sexing caused by the presence of polymorphism such as that identified in auklets (Dawson et al. 2001). 



References

Bruford MW, Hanotte O, Brookfield JFY, Burke T (1998) Multilocus and single-locus DNA fingerprinting. In: Molecular Genetic Analysis of Populations: A Practical Approach, 2nd edition, (ed. Hoelzel AR), pp. 287-336. IRL Press, Oxford. 



Dawson DA, Darby S, Hunter FM, Krupa AP, Jones IL and Burke T (2001) A critique of CHD-based molecular sexing protocols illustrated by a Z-chromosome polymorphism detected in auklets (Aves: Alcidae, Laridae). Molecular Ecology Notes, 1, 201-204.

Fridolfsson AK and Ellegren H (1999) A simple and universal method for molecular sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121.



Griffiths R, Double MC, Orr K and Dawson RJG (1998) A DNA test to sex most birds. Molecular Ecology, 7, 1071-1075.


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