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《Acta Agrestia Sinica》 2007-03
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Establishment and Optimization of ISSR Reaction System for Alfalfa

WANG Yu,YUAN Qing-hua (Institute of Animal Sciences,Chinse Academy of Agricultural Sciences,Beijing 100094,China)  
Alfalfa(Medicago sativa.L.) was subjected to ISSR(Inter-simple sequence repeats)-PCR to investigate the genetic diversity and the specific molecular markers linked to specific characters.By studying the main parameters we established the optimal ISSR-PCR reaction conditions in alfalfa.Results show that the optimum concentrations of seven reactants in 20 μL reaction mixture are as follows: 15 ng genomic DNA,0.2 mmol L-1 dNTP,0.4 μmol L-1 ISSR primer,0.8 U Taq DNA Polymerase,2 μL 10×PCR Buffer, 1.5 mmol L-1 MgCl2,2.5% deionized formamide.The suitable PCR procedure is one preliminary denaturation at 94℃ for 4 min;11 cycles each involved denaturation at 94℃ for 30 s,anneal at 62℃(62℃～58℃) for 45 s,with 1℃ declined each cycle,extended at 72℃ for 1 min 45 s;then followed by 24 cycles each with denaturation at 94℃ for 30 s,anneal at 52℃ for 45 s,extended at 72℃ for 1 min 45 s;and a final extension at 72℃ for 5 min,then keep the temperature at 25℃.
【Key Words】： Alfalfa ISSR PCR reaction system Optimization
【Fund】： 国家自然科学基金(30471230)
【CateGory Index】： S541.9
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