Nucleotidediversityandlinkagedisequilibriumat56stress-responseandphenologycandidategenesinEuropeanbeech(FagussylvaticaL.)

H.Lalagüe1,2,3,,K.Csilléry2,S.Oddou-Muratorio2,J.Safrana2,C.deQuattro3,B.Fady2,S.C.González-Martínez4,G.G.Vendramin3

1)ScuolaSuperioreSant'Anna,PiazzaMartiridellaLibertà33,56127Pisa,Italy

2)INRA,UR629EcologiedesForêtsMéditerranéennes(URFM),F-84914Avignon,France

3)PlantGeneticsInstitute,NationalResearchCouncil,ViaMadonnadelPiano10,50019SestoFiorentino(Firenze),Italy

4)DepartmentofForestEcologyandGenetics,NationalInstituteforAgricultureandFoodResearchandTechnology(INIA)-ForestResearchCentre(CIFOR),Madrid,Spain

Correspondingauthor:

G.G.Vendramin

CNR,PlantGeneticsInstitute,ViaMadonnadelPiano10,50019SestoFiorentino(Firenze),Italy

E-mail:

Tel:+390555225725

Fax:+390555225729

Appendix 2. Details of the PCR reaction mixture and the thermal profile for the amplification of the selected genes (see also Appendix 1). Step1: denaturation, step2: annealing, step3: extension. Temperature in Celsius degrees.

PCR protocol 1 / PCR protocol 2 / PCR protocol 3
DNA / 1µl (20ng/µl) / DNA / 1µl (20ng/µl) / DNA / 1µl (20ng/µl)
Buffer 5x GoTaq / 3µl / Buffer 5x GoTaq / 3µl / Buffer 5x GoTaq / 3µl
10mM dNTP / 0,3µl / 10mM dNTP / 0,3µl / 10mM dNTP / 0,3µl
10µM primers F / 0,3µl / 10µM primers F / 0,3µl / 10µM primers F / 0,3µl
10µM primers R / 0,3µl / 10µM primers R / 0,3µl / 10µM primers R / 0,3µl
TAQ (MgCl2 1,5mM) / 0,15µl / TAQ (MgCl2 1,5mM) / 0,15µl / TAQ (MgCl2 1,5mM) / 0,15µl
H2O / 9,95µl / H2O / 9,95µl / H2O / 9,95µl
Cycle 1 x1 / 94° 5 min / Cycle 1 x1 / 94° 5 min / Cycle 1 x1 / 94° 5 min
Cycle 2 x10 / step1 / 94° 30 sec / Cycle 3 x35 / step1 / 94° 30 sec / Cycle 3 x35 / step1 / 94° 30 sec
step2 / 65-55° 30s / step2 / 60° 30 sec / step2 / 63° 30 sec
step3 / 72° 1 min / step3 / 72° 1 min / step3 / 72° 1 min
Cycle 3 x35 / step1 / 94° 30 sec / Cycle 4 x1 / step1 / 72° 7 min / Cycle 4 x1 / step1 / 72° 7 min
step2 / 55° 30 sec / step2 / 7° ∞ / step2 / 7° ∞
step3 / 72° 1 min
Cycle 4 x1 / step1 / 72° 7 min
step2 / 7° ∞

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