аЯрЁБс>ўџ 46ўџџџ3џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅС5@ №П bjbjЯ2Я2 8 ­X­X џџџџџџˆДДДДДДДШ№№№№ ќШні (D D D D D D D \^^^^^^$гR%Œ‚ДК D D К К ‚ДДD D —ž ž ž К ZДD ДD \ž К \ž ž Д ДДД D  аї;Х№ RД \­0нД Бf .БД ШШДДДДД XБД PD  ф rž V \В D D D ‚‚ШШФŒd” ШШŒКљТЋгАвЕ Molecular Genetics Facility Bird sex-typing using genetic markers by Deborah Dawson (D.A.Dawson @КљТЋгАвЕ.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 КљТЋгАвЕ Molecular Genetics Facility is detailed here. DNA was extracted from blood following Bruford et al. (1998). Each 10-Еl reaction contained about 50 ng of genomic DNA, 1 ЕM 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:- 94АC 2 minutes  then:  94АC 45 seconds 48АC 45 seconds 72АC 45 seconds for 40 cycles, followed by: 72АC for 5 minutes The reaction profile used with the Fridolfsson and Ellegren (1999) 2550F/2718R primers was:- 94АC 2 minutes then:  94АC 30 seconds 60АC 30 seconds (reducing by 1АC each cycle),  72АC 30 seconds for 1 cycle,  94АC 30 seconds 59АC 30 seconds (reducing by 1АC each cycle),  72АC 30 seconds for 1 cycle,  etc, reducing annealing temperature by 1АC each cycle until.. 94АC 30 seconds  51АC 30 seconds (reducing by 1АC each cycle),  72АC 30 seconds for 1 cycle,  followed by: 94АC 30 seconds  50АC 30 seconds  72АC 30 seconds for 30 cycles.  followed by: 72АC 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: 94АC 2 min then 94АC 15 s, 50АC 20 s, 72АC 25 s for 40 cycles followed by 72АC 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 ( HYPERLINK "http://www.shef.ac.uk/misc/groups/molecol/Dawsonetal2001.pdf" 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.   HYPERLINK "http://www.shef.ac.uk/misc/groups/molecol/Dawsonetal2001.pdf" 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. &LM|ьђЁ В О Ф и њ " Y | … Ј X d і DФ bc­ЎЕЛРСЦаОП  єѕџ‰‹ћќ  їяътяозояозоЭоЭоЭоЭоЦоСоСоЙоЙГЊГЙояоЙоЙГЁГЙоЦоЦоhЇghр?Я0J5\hр?Я0J6] hр?Я0Jjhр?ЯU hр?Я>* hр?Я5\hр?ЯOJQJ^J hр?Я6]hр?Яhр?Яhр?Я\ hр?Я\hр?Я5>*\ha} hр?Я\1&L|Ё Д $ Њ  Ц і FЂФ"›ќ;ёіЖЦвОїђђээээээээээээээээээээээээgdр?Яgda} $a$gda}  ў–  њѕѓgdр?Яgdр?Я6&P 1F:pМgnАƒ. 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